Increasing plant growth by modulating omega-amidase expression in plants

ABSTRACT

The present disclosure relates to compositions and methods for increasing the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules in plants by modulating levels of Ω-amidase to increase nitrogen use efficiency, resulting in enhanced growth, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/037,307, filed Feb. 28, 2011, and which claims the benefit under 35USC 119(e) of U.S. Provisional Patent Application No. 61/308,971, filedFeb. 28, 2010, the disclosure of which is hereby incorporated byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.DE-AC52-06NA25396, awarded by the United States Department of Energy toLos Alamos National Security, LLC. The government has certain rights inthis invention.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“87485-946184-SEQLIST.txt” created Jun. 4, 2015, and containing 183,340bytes. The material contained in this text file is incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

As the human population increases worldwide, and available farmlandcontinues to be destroyed or otherwise compromised, the need for moreeffective and sustainable agriculture systems is of paramount interestto the human race. Improving crop yields, protein content, and plantgrowth rates represent major objectives in the development ofagriculture systems that can more effectively respond to the challengespresented.

In recent years, the importance of improved crop production technologieshas only increased as yields for many well-developed crops have tendedto plateau. Many agricultural activities are time sensitive, with costsand returns being dependent upon rapid turnover of crops or upon time tomarket. Therefore, rapid plant growth is an economically important goalfor many agricultural businesses that involve high-value crops such asgrains, vegetables, berries and other fruits.

Genetic engineering has and continues to play an increasingly importantyet controversial role in the development of sustainable agriculturetechnologies. A large number of genetically modified plants and relatedtechnologies have been developed in recent years, many of which are inwidespread use today (Factsheet: Genetically Modified Crops in theUnited States, Pew Initiative on Food and Biotechnology, August 2004).The adoption of transgenic plant varieties is now very substantial andis on the rise, with approximately 250 million acres planted withtransgenic plants in 2006.

While acceptance of transgenic plant technologies may be graduallyincreasing, particularly in the United States, Canada and Australia,many regions of the World remain slow to adopt genetically modifiedplants in agriculture, notably Europe. Therefore, consonant withpursuing the objectives of responsible and sustainable agriculture,there is a strong interest in the development of genetically engineeredplants that do not introduce toxins or other potentially problematicsubstances into plants and/or the environment. There is also a stronginterest in minimizing the cost of achieving objectives such asimproving herbicide tolerance, pest and disease resistance, and overallcrop yields. Accordingly, there remains a need for transgenic plantsthat can meet these objectives.

The goal of rapid plant growth has been pursued through numerous studiesof various plant regulatory systems, many of which remain incompletelyunderstood. In particular, the plant regulatory mechanisms thatcoordinate carbon and nitrogen metabolism are not fully elucidated.

These regulatory mechanisms are presumed to have a fundamental impact onplant growth and development.

The metabolism of carbon and nitrogen in photosynthetic organisms mustbe regulated in a coordinated manner to assure efficient use of plantresources and energy. Current understanding of carbon and nitrogenmetabolism includes details of certain steps and metabolic pathwayswhich are subsystems of larger systems. In photosynthetic organisms,carbon metabolism begins with CO₂ fixation, which proceeds via two majorprocesses, termed C-3 and C-4 metabolism. In plants with C-3 metabolism,the enzyme ribulose bisphosphate carboxylase (RuBisCo) catalyzes thecombination of CO₂ with ribulose bisphosphate to produce3-phosphoglycerate, a three carbon compound (C-3) that the plant uses tosynthesize carbon-containing compounds. In plants with C-4 metabolism,CO₂ is combined with phosphoenol pyruvate to form acids containing fourcarbons (C-4), in a reaction catalyzed by the enzyme phosphoenolpyruvate carboxylase. The acids are transferred to bundle sheath cells,where they are decarboxylated to release CO₂, which is then combinedwith ribulose bisphosphate in the same reaction employed by C-3 plants.

Numerous studies have found that various metabolites are important inplant regulation of nitrogen metabolism. These compounds include theorganic acid malate and the amino acids glutamate and glutamine.Nitrogen is assimilated by photosynthetic organisms via the action ofthe enzyme glutamine synthetase (GS) which catalyzes the combination ofammonia with glutamate to form glutamine. GS plays a key role in theassimilation of nitrogen in plants by catalyzing the addition ofammonium to glutamate to form glutamine in an ATP-dependent reaction(Miflin and Habash, 2002, Journal of Experimental Botany, Vol. 53, No.370, pp. 979-987). GS also reassimilates ammonia released as a result ofphotorespiration and the breakdown of proteins and nitrogen transportcompounds. GS enzymes may be divided into two general classes, onerepresenting the cytoplasmic form (GS1) and the other representing theplastidic (i.e., chloroplastic) form (GS2).

Previous work has demonstrated that increased expression levels of GS1result in increased levels of GS activity and plant growth, althoughreports are inconsistent. For example, Fuentes et al. reported that CaMVS35 promoter-driven overexpression of Alfalfa GS1 (cytoplasmic form) intobacco resulted in increased levels of GS expression and GS activity inleaf tissue, increased growth under nitrogen starvation, but no effecton growth under optimal nitrogen fertilization conditions (Fuentes etal., 2001, J. Exp. Botany 52: 1071-81). Temple et al. reported thattransgenic tobacco plants overexpressing the full length Alfalfa GS1coding sequence contained greatly elevated levels of GS transcript, andGS polypeptide which assembled into active enzyme, but did not reportphenotypic effects on growth (Temple et al., 1993, Molecular and GeneralGenetics 236: 315-325). Corruzi et al. have reported that transgenictobacco overexpressing a pea cytosolic GS1 transgene under the controlof the CaMV S35 promoter show increased GS activity, increased cytosolicGS protein, and improved growth characteristics (U.S. Pat. No.6,107,547). Unkefer et al. have more recently reported that transgenictobacco plants overexpressing the Alfalfa GS1 in foliar tissues, whichhad been screened for increased leaf-to-root GS activity followinggenetic segregation by selfing to achieve increased GS1 transgene copynumber, were found to produce increased 2-hydroxy-5-oxoproline levels intheir foliar portions, which was found to lead to markedly increasedgrowth rates over wild type tobacco plants (see, U.S. Pat. Nos.6,555,500; 6,593,275; and 6,831,040).

Unkefer et al. have further described the use of 2-hydroxy-5-oxoproline(also known as 2-oxoglutaramate) to improve plant growth (U.S. Pat. Nos.6,555,500; 6,593,275; 6,831,040). In particular, Unkefer et al. disclosethat increased concentrations of 2-hydroxy-5-oxoproline in foliartissues (relative to root tissues) trigger a cascade of events thatresult in increased plant growth characteristics. Unkefer et al.describe methods by which the foliar concentration of2-hydroxy-5-oxoproline may be increased in order to trigger increasedplant growth characteristics, specifically, by applying a solution of2-hydroxy-5-oxoproline directly to the foliar portions of the plant andover-expressing glutamine synthetase preferentially in leaf tissues.

SUMMARY OF THE INVENTION

The present disclosure is based on the surprising discovery thatincreasing Ω-amidase (omega-amidase) expression in the root tissues of aplant results in an increase in the plant's leaf-to-root ratio of thesignal metabolite 2-oxoglutaramate and related proline molecules.Additionally, increasing Ω-amidase expression in the root tissues of aplant results in decreased Ω-amidase expression in leaf tissue.Advantageously, modulating the expression of Ω-amidase in plants resultsin plants with increased nitrogen use efficiency, which in turn resultsin enhanced growth and other agronomic characteristics, including fastergrowth rates, greater seed and fruit/pod yields, earlier and moreproductive flowering, increased tolerance to high salt conditions, andincreased biomass yields.

Accordingly, one aspect of the present disclosure relates to atransgenic plant containing an Ω-amidase transgene, where the Ω-amidasetransgene is operably linked to a root-preferred promoter.

Another aspect of the present disclosure provides a transgenic planthaving inhibited expression of endogenous Ω-amidase in leaf tissue.

Still another aspect of the present disclosure relates to a method forincreasing nitrogen use efficiency of a plant relative to a wild type oruntransformed plant of the same species, by: (a) introducing anΩ-amidase transgene into the plant, where the Ω-amidase transgene isoperably linked to a root-preferred promoter; (b) expressing theΩ-amidase transgene in root tissue of the plant or the progeny of theplant; and (c) selecting a plant having an increased leaf-to-root ratioof 2-oxoglutaramate relative to a plant of the same species that doesnot contain an Ω-amidase transgene, where the increased leaf-to-rootratio of 2-oxoglutaramate results in increased nitrogen use efficiency.

Yet another aspect of the present disclosure relates to a method forincreasing nitrogen use efficiency of a plant relative to a wild type oruntransformed plant of the same species, by: (a) inhibiting endogenousΩ-amidase expression in leaf tissue of the plant; and (b) selecting aplant having an increased leaf-to-root ratio of 2-oxoglutaramaterelative to a plant of the same species that does not have inhibitedendogenous Ω-amidase expression in leaf tissue, where the increasedleaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen useefficiency

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of a metabolic pathway of nitrogenassimilation and 2-oxoglutaramate biosynthesis.

FIG. 2A-C depict amino acid sequence alignments of an Arabidopsisthaliana Ω-amidase with other putative plant Ω-amidases.

FIG. 3A-B depict amino acid sequence alignments of an Arabidopsisthaliana Ω-amidase with other putative animal Ω-amidases.

FIG. 4 graphically depicts a plot of plant fresh weight values plottedagainst 2-oxoglutaramate concentration.

FIG. 5 depicts a photograph comparing Ω-amidase transgenic and controlalfalfa plants.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; CurrentProtocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons,Inc. 2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed.,Humana Press, 1.sup.st edition, 2004); and, Agrobacterium Protocols(Wan, ed., Humana Press, 2.sup.nd edition, 2006). As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

Each document, reference, patent application or patent cited in thistext is expressly incorporated herein in its entirety by reference, andeach should be read and considered as part of this specification. Thatthe document, reference, patent application or patent cited in thisspecification is not repeated herein is merely for conciseness.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof (“polynucleotides”) in eithersingle- or double-stranded form.

Unless specifically limited, the term “polynucleotide” encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., 1991, NucleicAcid Res. 19: 5081; Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608;and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The term “promoter” refers to a nucleic acid control sequence orsequences that direct transcription of an operably linked nucleic acid.As used herein, a “plant promoter” is a promoter that functions inplants. Promoters include necessary nucleic acid sequences near thestart site of transcription, such as, in the case of a polymerase IItype promoter, a TATA element. As used herein, a promoter may includethe full nucleotide sequence, or may only include the core domain orsequence that directs transcription of the operably linked nucleic acid.A promoter also optionally includes distal enhancer or repressorelements, which can be located as much as several thousand base pairsfrom the start site of transcription. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is active underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an .alpha. carbonthat is bound to a hydrogen, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups (e.g., norleucine)or modified peptide backbones, but retain the same basic chemicalstructure as a naturally occurring amino acid. Amino acid mimeticsrefers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “plant” includes whole plants, plant organs (e.g., leaves,stems, flowers, roots, reproductive organs, embryos and parts thereof,etc.), seedlings, seeds and plant cells and progeny thereof. The classof plants which can be used in the method of the invention is generallyas broad as the class of higher plants amenable to transformationtechniques, including angiosperms (monocotyledonous and dicotyledonousplants), as well as gymnosperms. It includes plants of a variety ofploidy levels, including polyploid, diploid, haploid and hemizygous.

The terms “GPT polynucleotide” and “GPT nucleic acid” are usedinterchangeably herein, and refer to a full length or partial lengthpolynucleotide sequence of a gene which encodes a polypeptide involvedin catalyzing the synthesis of 2-oxoglutaramate, and includespolynucleotides containing both translated (coding) and un-translatedsequences, as well as the complements thereof. The term “GPT codingsequence” refers to the part of the gene which is transcribed andencodes a GPT protein. The term “targeting sequence” and “transitpeptide” are used interchangeably and refer to the amino terminal partof a protein which directs the protein into a subcellular compartment ofa cell, such as a chloroplast in a plant cell. GPT polynucleotides arefurther defined by their ability to hybridize under defined conditionsto the GPT polynucleotides specifically disclosed herein, or to PCRproducts derived therefrom.

A “GPT transgene” is a nucleic acid molecule comprising a GPTpolynucleotide which is exogenous to transgenic plant, or plant embryo,organ or seed, harboring the nucleic acid molecule, or which isexogenous to an ancestor plant, or plant embryo, organ or seed thereof,of a transgenic plant harboring the GPT polynucleotide. A “GPTtransgene” may encompass a polynucleotide that encodes either a fulllength GPT protein or a truncated GPT protein, including but not limitedto a GPT protein lacking a chloroplast transit peptide. Moreparticularly, the exogenous GPT transgene will be heterogeneous with anyGPT polynucleotide sequence present in wild-type plant, or plant embryo,organ or seed into which the GPT transgene is inserted. To this extentthe scope of the heterogeneity required need only be a single nucleotidedifference. However, preferably the heterogeneity will be in the orderof an identity between sequences selected from the following identities:0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.

The terms “GS polynucleotide” and “GS nucleic acid” are usedinterchangeably herein, and refer to a full length or partial lengthpolynucleotide sequence of a gene which encodes a glutamine synthetaseprotein, and includes polynucleotides containing both translated(coding) and un-translated sequences, as well as the complementsthereof. The term “GS coding sequence” refers to the part of the genewhich is transcribed and encodes a GS protein. The terms “GS1polynucleotide” and “GS1 nucleic acid” are used interchangeably herein,and refer to a full length or partial length polynucleotide sequence ofa gene which encodes a glutamine synthetase isoform 1 protein, andincludes polynucleotides containing both translated (coding) andun-translated sequences, as well as the complements thereof. The term“GS1 coding sequence” refers to the part of the gene which istranscribed and encodes a GS1 protein.

A “GS transgene” is a nucleic acid molecule comprising a GSpolynucleotide which is exogenous to transgenic plant, or plant embryo,organ or seed, harboring the nucleic acid molecule, or which isexogenous to an ancestor plant, or plant embryo, organ or seed thereof,of a transgenic plant harboring the GS polynucleotide. A “GS transgene”may encompass a polynucleotide that encodes either a full length GSprotein or a truncated GS protein, including but not limited to a GSprotein lacking a transit peptide. A “GS1 transgene” is a nucleic acidmolecule comprising a GS1 polynucleotide which is exogenous totransgenic plant, or plant embryo, organ or seed, harboring the nucleicacid molecule, or which is exogenous to an ancestor plant, or plantembryo, organ or seed thereof, of a transgenic plant harboring the GS1polynucleotide. A “GS1 transgene” may encompass a polynucleotide thatencodes either a full length GS protein or a truncated GS1 protein,including but not limited to a GS1 protein lacking a transit peptide.More particularly, the exogenous GS or GS1 transgene will beheterogeneous with any GS or GS1 polynucleotide sequence present inwild-type plant, or plant embryo, organ or seed into which the GS or GS1transgene is inserted. To this extent the scope of the heterogeneityrequired need only be a single nucleotide difference. However,preferably the heterogeneity will be in the order of an identity betweensequences selected from the following identities: 0.01%, 0.05%, 0.1%,0.5%, 1%, 5%, 10%, 15%, and 20%.

The terms “Ω-amidase (omega-amidase) polynucleotide” and “Ω-amidasenucleic acid” are used interchangeably herein, and refer to apolynucleotide sequence of a gene which encodes a polypeptide involvedin the enzymatic breakdown of 2-oxoglutaramate, and includespolynucleotides containing both translated (coding) and un-translatedsequences, as well as the complements thereof. The term “Ω-amidasecoding sequence” refers to the part of the gene which is transcribed andencodes an Ω-amidase protein. The Ω-amidase polynucleotides are furtherdefined by their ability to hybridize under defined conditions to theΩ-amidase polynucleotide specifically disclosed herein, or to PCRproducts derived therefrom.

An “Ω-amidase transgene” is a nucleic acid molecule comprising anΩ-amidase polynucleotide which is exogenous to transgenic plant, orplant embryo, organ or seed, harboring the nucleic acid molecule, orwhich is exogenous to an ancestor plant, or plant embryo, organ or seedthereof, of a transgenic plant harboring the Ω-amidase polynucleotide.An “Ω-amidase” may encompass a polynucleotide that encodes either a fulllength Ω-amidase protein or a truncated Ω-amidase protein, including butnot limited to an Ω-amidase protein lacking a chloroplast transitpeptide. More particularly, the exogenous Ω-amidase transgene will beheterogeneous with any Ω-amidase polynucleotide sequence present inwild-type plant, or plant embryo, organ or seed into which the Ω-amidasetransgene is inserted. To this extent the scope of the heterogeneityrequired need only be a single nucleotide difference. However,preferably the heterogeneity will be in the order of an identity betweensequences selected from the following identities: 0.01%, 0.05%, 0.1%,0.5%, 1%, 5%, 10%, 15%, and 20%.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3.sup.rd ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 25 to approximately 500 amino acids long. Typical domains aremade up of sections of lesser organization such as stretches of.beta.-sheet and .alpha.-helices. “Tertiary structure” refers to thecomplete three dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units.Anisotropic terms are also known as energy terms.

The term “isolated” refers to material which is substantially oressentially free from components which normally accompany the materialas it is found in its native or natural state. However, the term“isolated” is not intended refer to the components present in anelectrophoretic gel or other separation medium. An isolated component isfree from such separation media and in a form ready for use in anotherapplication or already in use in the new application/milieu. An“isolated” antibody is one that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, a nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a nucleic acid encoding aprotein from one source and a nucleic acid encoding a peptide sequencefrom another source. Similarly, a heterologous protein indicates thatthe protein comprises two or more subsequences that are not found in thesame relationship to each other in nature (e.g., a fusion protein).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95%identity) over a specified region, when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using a sequence comparison algorithms, or by manual alignmentand visual inspection. This definition also refers to the complement ofa test sequence, which has substantial sequence or subsequencecomplementarity when the test sequence has substantial identity to areference sequence. This definition also refers to the complement of atest sequence, which has substantial sequence or subsequencecomplementarity when the test sequence has substantial identity to areference sequence.

When percentage of sequence identity is used in reference topolypeptides, it is recognized that residue positions that are notidentical often differ by conservative amino acid substitutions, whereamino acids residues are substituted for other amino acid residues withsimilar chemical properties (e.g., charge or hydrophobicity) andtherefore do not change the functional properties of the polypeptide.Where sequences differ in conservative substitutions, the percentsequence identity may be adjusted upwards to correct for theconservative nature of the substitution.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from about 20to 600, usually about 50 to about 200, more usually about 100 to about150 in which a sequence may be compared to a reference sequence of thesame number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc.Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 are used, typically withthe default parameters described herein, to determine percent sequenceidentity for the nucleic acids and proteins of the invention. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measureof similarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a nucleic acid is considered similar to areference sequence if the smallest sum probability in a comparison ofthe test nucleic acid to the reference nucleic acid is less than about0.2, more preferably less than about 0.01, and most preferably less thanabout 0.001.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, highly stringent conditions are selected to be about5-10.degree. C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength pH. Low stringencyconditions are generally selected to be about 15-30.degree. C. below theTm. Tm is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0M sodium ion, typically about 0.01to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30.degree. C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60.degree. C. for long probes(e.g., greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal is at leasttwo times background, preferably 10 times background hybridization.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cased, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.

Genomic DNA or cDNA comprising GPT polynucleotides may be identified instandard Southern blots under stringent conditions using the GPTpolynucleotide sequences disclosed here. For this purpose, suitablestringent conditions for such hybridizations are those which include ahybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at37.degree. C., and at least one wash in 0.2.times.SSC at a temperatureof at least about 50.degree. C., usually about 55.degree. C. to about60.degree. C., for 20 minutes, or equivalent conditions. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions maybe utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantiallyidentical is if the reference sequence, amplified by a pair ofoligonucleotide primers, can then be used as a probe under stringenthybridization conditions to isolate the test sequence from a cDNA orgenomic library, or to identify the test sequence in, e.g., a northernor Southern blot.

Transgenic Plants with Altered Levels of Ω-Amidase Expression:

The present disclosure provides transgenic plants containing higherlevels of the signal metabolite 2-oxoglutaramate and its analogs infoliar tissues versus root or below-ground tissues and methods forincreasing nitrogen use efficiency of a plant generating plants.2-oxoglutaramate is a metabolite which is an extremely potent effectorof gene expression, metabolism and plant growth (U.S. Pat. No.6,555,500), and which may play a pivotal role in the coordination of thecarbon and nitrogen metabolism systems (Lancien et al., 2000, EnzymeRedundancy and the Importance of 2-Oxoglutarate in Higher PlantsAmmonium Assimilation, Plant Physiol. 123: 817-824).

The levels of 2-oxoglutaramate in leaf tissue may be increased byincreasing the biosynthesis of 2-oxoglutaramate. The biosynthesis of2-oxoglutaramte in leaf tissue may be preferentially increased to alevel sufficient to exceed the breakdown rate by, for example, bydecreasing the activity of the enzyme catalyzing the breakdown of2-oxoglutaramate (FIG. 1). Additionally, the levels of 2-oxoglutaramatein root tissue may be decreased by increasing the breakdown of2-oxoglutaramate. The breakdown rate of 2-oxoglutaramte in root tissuemay be preferentially increased by, for example, increasing the activityof the enzyme catalyzing the breakdown of 2-oxoglutaramate (FIG. 1).

The methods disclosed herein are used to generate transgenic plantsexhibiting higher leaf-to-root ratios of 2-oxoglutaramate when comparedto wild type or progenitor plants. In the practice of the disclosedmethods, 2-oxoglutaramate concentration in the leaf and root tissues maybe modulated by any one of or a combination of several approachesdisclosed herein. For example, the leaf-to-root ratio of 2 may beincreased by increasing the activity of Ω-amidases in root tissues or byinhibiting the activity of Ω-amidases in leaf tissues.

Modulation of the Ω-Amidase Pathway in Plants:

The present disclosure is based on the surprising discovery that theleaf-to-root ratio of 2-oxoglutaramate may be increased in plants bymodulating Ω-amidase expression in the root and/or leaf tissue ofplants. Moreover, increasing the leaf-to-root ratio of 2-oxoglutaramateresults in increased nitrogen use efficiency.

Applicants have identified an Ω-amidase pathway that may be modulated toachieve the objective of increasing the relative leaf to rootconcentration of 2-oxoglutaramate thereby triggering higher Nitrogenassimilation and carbon metabolism dynamics resulting in enhanced growthrates and agronomic characteristics. The only intermediate in thispathway, 2-oxoglutaramate, ostensibly functions as a signal metabolitethat reflects the flux of assimilated nitrogen. Increased levels of2-oxoglutaramate trigger a striking increase in resource acquisitionrates, carbon and nitrogen metabolism, and overall growth. Plantstreated with 2-oxoglutaramate or engineered to produce increased2-oxoglutaramate levels in leaf show greater leaf nitrogen and medianitrogen use efficiency. The resulting increase in overall growthmetabolism is accompanied by increased leaf-to-root ratios in2-oxoglutaramate pools, which are in turn controlled by glutaminesynthetase, glutamine phenylpyruvate transaminase and Ω-amidaseactivities, as well as by the availability of nitrogen, in a complex,interrelated and tissue-specific manner. Moreover, increasing theleaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen useefficiency. As used herein, “increased nitrogen use efficiency” and“increasing nitrogen use efficiency” refers to plants that have enhancedgrowth and better agronomic characteristics. Agronomic characteristicsinclude, without limitation, faster growth rates, greater seed andfruit/pod yields, earlier and more productive flowering, increasedtolerance to high salt conditions, and/or increased biomass yieldsresulting from increased nitrogen utilization mediated by an increasedleaf-to-root ratio of 2-oxoglutaramate.

As described in Example 1, infra, transgenic plants engineered toover-express GS and/or GPT show lower Ω-amidase activities in the leavesand greater Ω-amidase activity in the roots. GPT and GS+GPT transgenicplants showed the largest increases in root Ω-amidase activity. Theseplants responded to expression of the transgenes by altering theirΩ-amidase activities such that they tend to increase the leaf2-oxoglutaramate pool and maintain the root 2-oxoglutaramate pool. Theseresponses combined in the GS+GPT over-expressing plants to generate thehighest leaf and lowest root 2-oxoglutaramate pools and the highest leafand lowest GS and GPT activities.

Without wishing to be bound by theory, it is believed that the signalmetabolite, 2-oxoglutaramate, provides two different messages, dependingupon the tissue. Increased 2-oxoglutaramate in the leaves is stimulatory(Tables 2-4) and appears to effectively convey the message that nitrogenis abundant and carbon must be fixed to take advantage of the increasednitrogen. The increased nitrogen supply-driven faster growth isaccompanied by an increase in leaf-to-root 2-oxoglutaramate pools. Theincrease in the leaf 2-oxoglutaramate pool is apparently key to thestimulation. Without wishing to be bound by theory, it is believed thatthe plant's strategy to maintain near normal or diminished2-oxoglutaramate pool in the roots is a mechanism they may be using toovercome the apparent contradiction of how plants grow faster whenfertilized with nitrogen and yet display the long-observed inhibition ofnitrate uptake and assimilation in roots by a “N metabolite downstreamof NH₃” (Foyer et al., 2002, Kluwer Academic Publishers, TheNetherlands). If the inhibitory nitrogen metabolite is 2-oxoglutaramate,then the mechanism could be to manage its concentration downward whennitrogen is abundant to let the plant prosper and, conversely keep2-oxoglutaramate higher when nitrogen is scarce and the plant tries toconserve a limiting resource to survive to reproduce.

Without wishing to be bound by theory, it is believed that the resultsdisclosed herein indicate that modulating Ω-amidase activity in plantsis useful in driving increased levels of 2-oxoglutaramate in leavesrelative to roots. Two approaches are hereinafter described: (1)promoting the breakdown of 2-oxoglutaramate by upregulating Ω-amidaseactivity in root tissues, and (2) impairing the breakdown of2-oxoglutaramate in leaf tissues by impairing, downregulating, ordeactivating leaf Ω-amidase activity.

Promoting the Breakdown of 2-Oxoglutaramate by Upregulating Ω-AmidaseActivity in Root Tissues:

Certain aspects of the present disclosure relate to transgenic plantscontaining an Ω-amidase transgene, where the Ω-amidase transgene isoperably linked to a root-preferred promoter.

It has been previously shown that an Ω-amidase-like pathway in plantsthat is possibly involved in 2-oxoglutaramate breakdown (Von GusgtavSchwab 1936, Planta Archiv fur wissenschaftliche botanik. 25.Band, 4.Heft. p 579-606. [German publication]; Olenicheva, LS1955, Biokhimiia20(2):165-172. [Russian publication]; and Yamamoto, Y. 1955, Journal ofBiochemistry 42:763-774). However, none of these studies identified anΩ-amidase protein.

Accordingly, it is believed that that an Ω-amidase pathway is involvedin the breakdown of 2-oxoglutaramate and its analogs in plants (FIG. 1).Additionally, an Ω-amidase enzyme has been shown to be capable ofcatalyzing the breakdown of 2-oxoglutaramate in animal cells by openingthe ring of 2-oxoglutaramate and removing the nitrogen to yield a ketoacid (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry,pages 281-303; Meister, 1952, J. Biochem. 197: 304).

Applicants have identified a putative plant Ω-amidase gene and protein(Arabidopsis thaliana gene AT5g12040, F14F18 210 mRNA). Theidentification of the putative plant Ω-amidase was based on sequencehomology analysis to Ω-amidase gene sequences from other organismsincluding human and rat. The nucleotide coding and translated amino acidsequences thereof are shown below.

Arabidopsis ω-omega.-amidase nucleotide coding sequence (Genbankaccessions AY075592.1 with GI 19715573 corresponding to protein NP445766(full-length protein AAL91613.1=SEQ ID NO:46)) [SEQ ID NO: 2]:

AAAGTGAAATGAAGTCAGCAATTTCATCGTCACTCTTCTTCAATTCGAAGAATCTTTTAAACCCTAATCCTCTTTCTCGCTTCATTTCTCTCAAATCTAACTTCCTCCCTAAATTATCTCCGAGATCGATCACTAGTCACACCTTGAAGCTCCCATCTTCGTCAACCTCAGCTTTAAGATCCATTTCCTCTTCCATGGCTTCTTCTTTCAACCCTGAACAAGCTAGAGTTCCCTCTGCTCTTCCTCTCCCAGCTCCTCCGTTGACCAAATTCAACATCGGATTGTGTCAGCTATCTGTTACATCTGACAAAAAGAGAAACATCTCTCATGCTAAAAAAGCCATTGAAGAAGCTGCTTCTAAAGGAGCTAAGCTTGTTCTCTTACCCGAAATTTGGAACAGTCCGTATTCCAATGATAGTTTTCCAGTTTATGCGGAGGAGATTGATGCAGGTGGTGATGCTTCTCCTTCAACGGCAATGCTTTCTGAAGTTTCCAAACGTCTCAAGATTACAATCATTGGTGGATCTATACCAGAAAGAGTTGGAGATCGTTTGTATAACACTTGCTGTGTCTTTGGTTCCGATGGAGAGCTAAAAGCTAAGCATCGGAAGATACATTTATTTGATATAGACATTCCCGGGAAGATTACTTTTATGGAATCCAAAACTCTTACTGCTGGAGAGACACCAACAATCGTTGACACAGATGTAGGGCGTATTGGAATAGGCATCTGTTATGATATCAGGTTCCAGGAGTTAGCTATGATATATGCTGCAAGAGGGGCTCATTTGCTGTGCTACCCGGGAGCCTTTAACATGACAACTGGACCATTGCATTGGGAATTACTACAAAGGGCCAGGGCTACGGATAATCAGTTATATGTGGCGACATGCTCACCTGCCAGAGATTCAGGAGCTGGCTACACTGCTTGGGGGCACTCAACACTCGTTGGGCCTTTTGGAGAAGTACTAGCAACGACTGAGCATGAGGAGGCCATTATCATAGCAGAGATTGATTACTCTATCCTTGAACAACGAAGGACTAGCCTTCCATTGAATAGGCAGCGGCGGGGAGATCTTTACCAGCTTGTAGACGTACAGCGCTTAGACTCTAAATGAACGCAGCAGTAACTGTATATCTGAGAGATATTGCGAGTTGAGCACGATTTGGTTACTTACAACTTCATGCATGATCAGTCATTTCTCCACAACTTTGCTGAGATATGTAAAAGAATAAAAATCAAACTTTTGAGTTAAAATCGAACAAAGGCAAGTAAATTCTGCTTAGATAATGTGAACTCCACCCACTTGCCATGTGTTTGTTGTTTATAAACTTCAATGCATTCTGATAACG

Mature Arabidopsis ω-amidase amino acid sequence; and derived from thetranslation product of SEQ ID NO: 2, above (Genbank accessionAAL91613.1) [SEQ ID NO: 3]:

MASSFNPEQARVPSALPLPAPPLTKFNIGLCQLSVTSDKKRNISHAKKAIEEAASKGAKLVLLPEIWNSPYSNDSFPVYAEEIDAGGDASPSTAMLSEVSKRLKITIIGGSIPERVGDRLYNTCCVFGSDGELKAKHRKIHLFDIDIPGKITFMESKTLTAGETPTIVDTDVGRIGIGICYDIRFQELAMIYAARGAHLLCYPGAFNMTTGPLHWELLQRARATDNQLYVATCSPARDSGAGYTAWGHSTLVGPFGEVLATTEHEEAIIIAEIDYSILEQRRTSLPLNRQRRGD LYQLVDVQRLDSK

Based on initial BLAST analysis in Genbank, the Arabidopsis Ω-amidasehas homologs in other plant species, as well as in bacteria, fungi,frogs, fish, and mammal. None of the identified homologs were annotatedas Ω-amidases. However, without wishing to be bound by theory, it isbelieved that these sequences also encode Ω-amidases. The amino acidsequences of the identified putative Ω-amidases are shown below.

Vitis vinifera amino acid sequence (Genbank accession XP 002279687.1)[SEQ ID NO: 4]:

MKSAALSALLSSTLSYASPPHLNLLRPATAVLCRSLLPTSTPNPFHTQLRTAKISASMSSSFKPEQARVPPAIPPPTPPLSKFKIGLCQLSVTADKERNIAHARKAIEEAVEKGAQLVLLPEIWNSPYSNDSFPVYAEDIDAGSDASPSTAMLSEVSHALKITIVGGSIPERCGDQLYNTCCVFGSDGKLKAKHRKIHLFDINIPGKITFMESKTLTAGGSPTIVDTEVGRIGIGICYDIRFSELAMLYAARGAHLICYPGAFNMTTGPLHWELLQRARAADNQLYVATCSPARDAGAGYVAWGHSTLVGPFGEVLATTEHEEAIIISEIDYSLIELRRTNLP LLNQRRGDLYQLVDVQRLDSQ

Zea mays amino acid sequence (Genbank accession ACN30911.1) [SEQ ID NO:5]:

MVAAAAAAAAATATAAALLAPGLKLCAGRARVSSPSGLPLRRVTAMASAPNSSFRPEEARSPPALELPIPPLSKFKVALCQLSVTADKSRNIAHARAAIEKAASDGAKLVVLPEIWNGPYSNDSFPEYAEDIEAGGDAAPSFSMLSEVARSLQITLVGGSIAERSGNNLYNTCCVFGSDGQLKGKHRKIHLFDIDIPGKITFKESKTLTAGQSPTVVDTDVGRIGIGICYDIRFQELAMLYAARGAHLLCYPGAFNMTTGPLHWELLQRARAADNQLFVATCAPARDTSAGYVAWGHSTLVGPFGEVIATTEHEEATIIADIDYSLIEQRRQFLPLQHQRRGD LYQLVDVQRLGSQ

Populus trichocarpa amino acid sequence (Genbank accession XP002309478.1) [SEQ ID NO: 6]:

MKSAISSTTTLLSSKNLSLKLHLNHSPLSRLPSSLFRSKSNTHFPSLLPRNNSTHNQKSQIHTPIMASSFMPEQARAPPALPLPVPPFKIGLCQLSVTADKERNIAHARKAIEEAAAKGAKLVMLPEIWNSPYSNDCFPVYAEDIDAGGEASPSTAMLSEAAGLLKVTIVGGSIPERSGDRLYNTCCVFDSDGKLKAKHRKIHLFDIDIPGKITFIESKTLTAGETPTIVDTEVGRIGIGICYDIRFQELAIIYAARGAHLICYPGAFNMTTGPLHWELLQRARAADNQLYVATCSPARDVAAGYVAWGHSTLVGPFGEVLATTEHEEDIIIAEIDYSLLEVRRTNLPLTKQRRGDLYQLVDVQRLKSDS

Picea sitchensis amino acid sequence (Genbank accession ABK22312.1) [SEQID NO: 7]:

MTPLLSYSLRVVASALRPKSSIASAVGRLSATPKRFPANRLRISYRNYNAAMAKPEDARSPPALPLPSAPNGGKFKIALCQLSVTENKERNIAHARDAIEAAADNGAQLVVLPEIWNGPYSNASFPVYAEDIDAGGSASPSTSMLSEVARSKGITIVGGSISERSGDHLYNTCCIFGKDGELKAKHRKIHLFDIDIPGKISFMESKTLTAGNTPTIVDTDVGRIGIGICYDIRFQELAMLYAARGAHLICYPGAFNMTTGPLHWELLQRARAIDNQLYVATCSPARDINAGYVAWGHSTLVAPFGEIVATTEHEEATVIADIDYSRIEERRMNMPLEKQRHGD LYQLVDVSRLDTAKH

Oryza sativa amino acid sequence (Genbank accession NP_(—)001049134.1)[SEQ ID NO: 8]:

MATAASFRPEAARSPPAVQPPAPPLSKFKVALCQLSVTADKARNIARAREAIEAAAAGGAKLVLLPEIWNGPYSNDSFPEYAEDIEAGGDAAPSFSMMSEVARSLQITLVGGSISERSGNKLYNTCCVFGSDGELKGKHRKIHLFDIDIPGKITFKESKTLTAGQDLTVVDTDVGRIGIGICYDIRFQELAMLYAARGAHLLCYPGAFNMTTGPLHWELLQRARAADNQLFVATCAPARDTSAGYIAWGHSTLVGPFGEVIATAEHEETTIMAEIDYSLIDQRRQFLPLQYQRR GDLYQLVDVQRSGSDE

Sorghum bicolor amino acid sequence (Genbank accessionXP_(—)002468410.1) [SEQ ID NO: 9]:

MRAAAAAAATSTAAALLAPGLKLCAGRARVSSCRLPLRRVAAMASAPNSSFRPEEARSPPALELPTPPLSKFKVALCQLSVTADKSRNIAHARAAIEKAASDGAKLVLLPEIWNGPYSNDSFPEYAEDIEAGGDAAPSFSMMSEVARSLQITLVDGQLKGKHRKIHLFDIDIPGKITFKESKTLTAGQSPTVVDTDVGRIGIGICYDIRFQELAMLYAARGAHLLCYPGAFNMTTGPLHWELLQRARQPAVCCNVRSSSRYQCRLCCLGTLHACWTFWRGDCNN

Ricinus communis amino acid sequence (Genbank accessionXP_(—)002516116.1) [SEQ ID NO: 10]:

MSASFNPEQARSPPALPLPTPPLTKAQFLLTSYLTILIYMIFKIGLCQLLVTPDKAKNIAHARKAIEEAAAKGAKLVLLPEIWNSPYSNDSFPVYAEDIDAGHVASPSTAMLSQLARLLNITIVGGSIPERSGDRLYNTCCVFDTQGNLIAKHRKIHLFDIDIPGKITFIESKTLTAGETPNIVDTEVGRIGIGICYDIRFQELAVLYAARGAHLICYPGAFNMTTGPLHWELLQRARAADNQLYVATCSPARDVGAGYVAWGHSTLVGPFGEILATTEHEQDIIIAEIDYSLIELRSQLSTTHLPLPTPTTTRDSTIEEEDDLVYIYI

Physcomitrella patens subsp. patens amino acid sequence (Genbankaccession XP_(—)001766085.1) [SEQ ID NO: 11]:

MASDFQPHMARQPPSESLPNAPNGGKYKLAVCQLSVTSDKAANIAHARQKIEAAADSGAQLIVLPEMWNCPYSNDSFPTYAEDIDAGLEASPSSHMLSEVARKKKVTIVGGSIPERNDGKLYNTCCVFDKNGELKAKFRKIHLFDIDIPGKITFKESDTLTPGEGLCVVDTDVGRIAVGICYDIRFPEMAMLYSARGAHIICYPGAFNMTTGPLHWELLQKARAVDNQIFVVTCSPARDTEAGYIAWGHSTVVGPFGEILATTEHEEATIFADIDYSQLDTRRQNMPLESQRR GDLYHLIDVTRKDTVKSS

Selaginella moellendorffii amino acid sequence (Genbank accessionXP_(—)002969787.1) [SEQ ID NO: 12]:

MPSSRYFWFLWQFKLAVCQLSICADKEQNIRHAREAIQTAADGGSKLVLLPEMWNCPYSNASFPIYAEDIDAGDSPSSKMLSDMAKSKEVTIIGGSIPERSGNHLYNTCCIYGKDGSLKGKHRKVHLFDIDIPGKIQFKESDTLTPGDKYTVVDTDVGRIGVGICYDIRFPEMAMTYAARGVHMICYPGAFNMTTGPAHWELLQKARAVDNQLFVATCSPARNPSAGYVAWGHSSVIGPFGEILASTGREEAIFYADIDYAQIKERRMNMPLDHQRRGDLYQLVDLTFTT

Medicago truncatula amino acid sequence (Genbank accession ACJ85250.1)[SEQ ID NO: 13]:

MAASSINSELARSPPAIPLPTPPLTNFKIGLCQLSVTSDKDKNIAHARTAIQDAAAKGAKLILLPEIWNSPYSNDSFPVYAEDIDAGGDASPSTAMLSELSSLLKITIVGGSIPERSGDRLYNTCCVFGTDGKLKAKHRKIHLFDIDIPGKITFIESLTLTAGDTPTIVDTEVGRIGIGICYDIRFPELAMIYAARGAHLLCYPGAFNMTTGPLHWELLQRARATDNQLYVATCSPARDTTGWLCGLGVTPLLLVLLEKFWLLQNARRQPL

Chlorella variabilis amino acid sequence (Genbank accession EFN54567.1)[SEQ ID NO: 14]:

MQALAKGMALVGVAGLSAAAGRRAACLRPLSSYTSATADVIDPPPPQKVPPPLPCCRCRHCCHRLASNQQLARPLLAGPSAQIKVALCQLAVGADKQANLTTARSAIEEAATAGADLVVLPEMWNCPYSNDSFPTYAEDVEAGDSPSTSMLSAAAAANRVVLVGGSIPERANGGRLYNTCFVYGRDGRLLGRHRKVHLFDIDIPGKITFKESLTLTPGEGLTVVGRLGIGICYDIRFPELALLYAARGVQLIVYPGAFNMTTGPVHWELLQRARAVDGQLFVATCSPARSEGTGYIAWGHSTAVGPFAEVLATTDEKAGIVYCHMDFAQLGERRANMPLRHQKRADLYSLLDLTRPNSLSNAGLHNGPVQRTLAGSSGIVGSGITRQLLMEGAKVVALLRKVDQKAGLLRDCQGAPIENLYPAVVEDVSKEEQCAAFVHEVVEQHGAIDHAVSCFGAWWQGGLLTEQSYAEFSRVLANFAGSHFTFVKYILPAMRQSHTSSMLFVTGGVGKRVLSADSGLATVGGAALYGIVRAAQAQYQGRPPRINELRIFALVTRHGEMPRSHSSIVEGLRAHSNRKVGNLAAEALAAAADDELLEVTSERLDGVMLMVGD

Volvox carteri f. nagariensis amino acid sequence (Genbank accessionXP_(—)002948137.1) [SEQ ID NO: 15]:

MHVTADKAQNLQTAKRAIEDAAAQGAKLVVLPEMWNCPYSNDSFPTYAEDIEGGASGSVAMLSAAAAAACVTLVAGSIPERCGDRLYNTCCVFNSRGELLAKHRKVHLFDIDIPGKITFKESLTLSPGPGPTVVDTEAGRLGIGICYDIRFPELAQLYAARGCQVLIYPGAFNMTTGPVHWELLARARAVDNQIFVITCSPARNPSSSYQAWGHSTVVGPFAEILATTDHQPGTIYTELDYSQLAERRANMPLRQQKRHDLYVLLDKTA

Chlamydomonas reinhardtii amino acid sequence (Genbank accessionXP_(—)001690839.1) [SEQ ID NO: 19]:

KVALCQLHVTADKEQNLRTARKAIEDAAAAGAKLVVLPEMFNCPYSNDSFPTYAEDIEGGASGSVAALSAAAAAARVTLVAGSIPERCQGKLYNTCCVFDSSGKLLAKHRKVHLFDIDIPGKITFKESLTLSPGPGPTVVDTEAGRLGIGICYDIRFPELAQIYAARGCQVLIYPGAFNMTTGPVHWELLAKARAVDNQVFVLTCSPARNPDSSYQAWGHSTALGPFAEVLATTEHSPATVFAELDYAQLDERRAAMPLRQQKRHDLYLLLDKTA

Micromonas pusilla CCMP1545 amino acid sequence (Genbank accessionXP_(—)003064056.1) [SEQ ID NO: 20]:

MRATKTTAAAAAAAAASSSGAGAPVPFARVPAPWSASGASASDAATPTPTPAPRVVKVALCOLACPTADKVANIARAREAIRNAAEGGAALVVLPEMWNCPYANESFPAHAETIGANDPTPSVTMLSEAAAAHDIVLVGGSIPERGVGVGGGGGADEEDVLYNACCVFDGKRGLIARHRKTHLFDVDIPGEISFRESDTLTEGEGLTVVDTAVGRVGVGICFDVRFGEMAAAMANRGADVLIYPGAFNTVTGPHHWELLQRARAVDNQARSIHWSPYDRCFVLTCSPARNTTGEGYQAWGHSTAVGPFAEVLATTDERPGIVFADLDLGEVTRRRRNMPLATQ RRGDLYALHDLGAVRGDA

Ectocarpus siliculosus amino acid sequence (Genbank accession CBJ25483)[SEQ ID NO: 21]:

MFLAAARRASPILLSLAVKTSTTAAFCSPRLANARTNTAAGATRTAYAACSISRNISLLSRPLSSMSASGASEGATAGAGSRRFVVAACQILCGSDKLANIATAESAVRDAAAAGAQVVVLPECWNGPYDTASFPVYAEPVPDPQGDETAADMPSAEQSPSAAMLCRAAAENKVWLVGGSVPEAGKDGGVYNTCIVVGPSGRIVAKHRKVHLFDIDVPGGITFKESDTLSPGDSITTVETPFGTIGVGICYDMRFPELSMAMRAAGSVLLCFPGAFNMTTGPAHWELLQRARALMDNQCFVVTASPARNPDSKYQAWGHSSIVDPWGTVVATTEHEEALVAEVDVGRVAEVRTSIPVSLQKRPDLYRLELP

Phaeodactylum tricornutum CCAP 1 055/1 amino acid sequence (Genbankaccession XP_(—)002183613.1) [SEQ ID NO: 22]:

MSASRQNDDDDDDDPSVLRVALCQLPVTNDKAQNHQTAREYLNRAANQGARLVVLPEIWNSPYATAAFPEYAEQLPDVLAQDGDGHTGVYESPSADLLRESAKEHKLWIVGGSIPERDDDDKIYNTSLVFDPQGNLVAKHRKMHLFDIDVPGGITFFESDTLSPGNTVSHFATPWGNIGLGICYDIRFPEYAMLLAKEHDCGILIYPGAFNLTTGPAHWELLQRGRAVDNQCFVLTASPARTEPPSKAGLYPHYTAWGHSTAVSPWGEVIATTNEKAGIVFADLDLSKVTEMRT SIPIGKQKRTDLYQLVGKS

Schizosaccharomyces pombe 972h amino acid sequence (Genbank accessionNP_(—)594154.1) [SEQ ID NO: 23]:

MNSKFFGLVQKGTRSFFPSLNFCYTRNIMSVSASSLVPKDFRAFRIGLVQLANTKDKSENLQLARLKVLEAAKNGSNVIVLPEIFNSPYGTGYFNQYAEPIEESSPSYQALSSMAKDTKTYLFGGSIPERKDGKLYNTAMVFDPSGKLIAVHRKIHLFDIDIPGGVSFRESDSLSPGDAMTMVDTEYGKFGLGICYDIRFPELAMIAARNGCSVMIYPGAFNLSTGPLHWELLARARAVDNEMFVACCAPARDMNADYHSWGHSTVVDPFGKVIATTDEKPSIVYADIDPSVMSTARNSVPIYTQRRFDVYSEVLPALKKEE

Aspergillus oryzae RIB40 amino acid sequence (Genbank accessionXP_(—)001819629.1) [SEQ ID NO: 24]:

MAALLKQPLKLALVQLASGADKAVNLAHARTKVLEAAQAGAKLIVLPECFNSPYGTQYFPKYAETLLPSPPTEDQSPSYHALSAIAAEAKAYLVGGSIPELEPTTKKYYNTSLVFSPTGSLIGTHRKTHLFDIDIPGKITFKESEVLSPGNQLTIVDLPDYGKIGLAICYDIRFPEAAMIAARKGAFALIYPGAFNMTTGPMHWSLLARARAVDNQLYVGLCSPARDMEATYHAWGHSLIANPAAEVLVEAEDKETIVYADLDNDTIQSTRKGIPVYTQRRFDLYPDVSAEK

Neurospora crassa OR74A amino acid sequence (Genbank accessionXP_(—)960906.1) [SEQ ID NO: 25]:

MASSTKHPILLKKPVKLACIQLASGADKSANLSHAADKVREAASGGANIVVLPECFNSPYGCDFFPSYAEQLLPSPPTVEQSPSFHALSAMARDNGIYLVGGSIPELAIEEGTEDKKTYYNTSLVFGPDGKLLASHRKVHLFDIDIPGKIKFKESDVLSPGNSVTLVDLPDYGRIAVAICYDIRFPELAMIAARKGCFALVYPGAFNTTTGPLHWRLQGQARAMDNQIYVALCSPARDISASYHAYGHSLIVDPMARVLVEAEESETIVSAELDGTKIEEARSGIPLRDQRRF DIYPDVSQAKPFF

Rhizobium leguminosarum by. viciae 3841 amino acid sequence (Genbankaccession YP_(—)769862.1) [SEQ ID NO: 26]:

MSFKAAAVQMCSGVDPVRNAAAMARLVREAAGQGAIYVQTPEMTGMLQRDRAAARAVLADEAHDIIVKTGSDLARELGIHMHVGSTAIALADGKIANRGFLFGPDGRILNRYDKIHMFDVDLDNGESWRESAAYTAGSEARVLSLPFAEMGFAICYDVRFPALFRAQAMAGAEVMTVPAAFTKQTGEAHWEILLRARAIENGVFVIAAAQAGRHEDGRESFGHSMIIDPWGTVLASAGATGEAVIVAEIDPSAVKAAHDKIPNLRNGREFSVEKIAGAIAGGVAA

Rhizobium etli CFN 42 amino acid sequence (Genbank accessionYP_(—)471237.1) [SEQ ID NO: 27]:

MSFKAAAIQMCSGVDPVKNAASMARLVREAAAQGATYVQTPEMTGMLQRDRAAARAVLADEAHDIIVKTGSELARELGIHVHVGSTAIALSDGKIANRGFLFGPDGRILNRYDKIHMFDVDLDNGESWRESAAYTAGSEARVLSLPFAEMGFAICYDVRFPALFRAQAVAGAEVMTVPSSFSRQTGEAHWEILLRARAIENGVFVIAAAQAGRHEDGRETFGHSIIIDPWGTVLASAGATGEAVILAEIDPGAVKAAHDKIPNLRDGREFSVEKIAGAVAGGVAA

Rhizobium leguminosarum by. trifolii WSM1325 amino acid sequence(Genbank accession YP_(—)002977603.1) [SEQ ID NO: 28]:

MSFKAAAVQMCSGVDPVKNAAAMARLVREAAGQGATYVQTPEMTGMLORDRTAARAVLADEAHDIIVKTGSELAIELGIHMHVGSTAIALADGKIANRGFLFGPDGRVLNRYDKIHMFDVDLDNGESWRESAAYTAGSEARVLSLPFAEMGFAICYDVRFPALFCAQAVAGAEVMTVPAAFTKQTGEAHWEILLRARAIENGVFVIAAAQAGRHEDGRETFGHSMIIDPWGTVLASAGATGEAVIVAEIDPAAVKAAHDKIPNLRNGREFSVEKIAGAIAGGVAA

Bradyrhizobium sp. ORS278 amino acid sequence (Genbank accessionYP_(—)001202760.1) [SEQ ID NO: 29]:

MSNDRSFTAAMVQMRTALLPEPSLEQGTRLIREAVAQGAQYVQTPEVSNMMQLNRTALFEQLKSEEEDPSLKAYRALAKELNIHLHIGSLALRFSAEKAVNRSFLIGPDGQVLASYDKIHMFDIDLPGGESYRESANYQPGETAVISDLPWGRLGLTICYDVRFPALYRALAESGASFISVPSAFTRKTGEAHWHTLLRARAIETGCFVFAAAQCGLHENKRETFGHSLIIDPWGEILAEGGVEPGVILARIDPSRVESVRQTIPSLQHGRRFGIADPKGGPDYLHLVRGSA

Sinorhizobium meliloti BL225C amino acid sequence (Genbank accessionZP_(—)07592670.1) [SEQ ID NO: 30]:

MPSSRYFWFLWQFKLAVCQLSICADKEQNIRHAREAIQTAADGGSKLVLLPEMWNCPYSNASFPIYAEDIDAGDSPSSKMLSDMAKSKEVTIIGGSIPERSGNHLYNTCCIYGKDGSLKGKHRKVHLFDIDIPGKIQFKESDTLTPGDKYTVVDTDVGRIGVGICYDIRFPEMAMTYAARGVHMICYPGAFNMTTGPAHWELLQKARAVDNQLFVATCSPARNPSAGYVAWGHSSVIGPFGEILASTGREEAIFYADIDYAQIKERRMNMPLDHQRRGDLYQLVDLTFTT

Sinorhizobium meliloti 1021 amino acid sequence (Genbank accessionNP_(—)386723.1) [SEQ ID NO: 31]:

MTFKAAAVQICSGVDPAGNAETMAKLVREAASRGATYVQTPEMTGAVQRDRTGLRSVLKDGENDVVVREASRLARELGIYLHVGSTPIARADGKIANRGFLFGPDGAKICDYDKIHMFDVDLENGESWRESAAYHPGNTARTADLPFGKLGFSICYDVRFPELFRQQAVAGAEIMSVPAAFTRQTGEAHWEILLRARAIENGLFVIAAAQAGTHEDGRETFGHSMIVDPWGRVLAEAGATGEEIIVAEIDVAAVHAARAKIPNLRNARSFVLDEVVPVGK GGAAA

Phytophthora infestans T30-4 amino acid sequence (Genbank accessionXP_(—)002999170.1) [SEQ ID NO: 32]:

MLGRTIRSQARHLRSPFLRLSSPMSTTAPKFKLALCQIAVGDDKQKNIATATAAVTEAAQNAAQVVSLPECWNSPYATTSFPQYAEEIPEKKAALNEKEHPSTFALSQLAAKLQIFLVGGSIPEKDATGKVYNTSVIFSPEGEILGKHRKVHLFDIDVPGKITFKESDTLSPGNSMTLFDTPYGKMGVGICYDIRFPELSMLMKKQGAKVLLFPGAFNLTTGPAHWELLQRARAVDNQLYVAATSPARGPEGGYQAWGHSTVISPWGEVVATCGHGESIVYAEVDLEKVEEMRR NIPTTNQTRSDLYELVQK

Homo sapiens amino acid sequence (Genbank accession NP_(—)064587.1) [SEQID NO: 33]:

MTSFRLALIQLQISSIKSDNVTRACSFIREAATQGAKIVSLPECFNSPYGAKYFPEYAEKIPGESTQKLSEVAKECSIYLIGGSIPEEDAGKLYNTCAVFGPDGTLLAKYRKIHLFDIDVPGKITFQESKTLSPGDSFSTFDTPYCRVGLGICYDMRFAELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRSRAVDNQVYVATASPARDDKASYVAWGHSTVVNPWGEVLAKAGTEEAIVYSDIDLKKLAEIRQQIPVFRQKRSDLYAVEMKKP

Equus caballus amino acid sequence (Genbank accession XP_(—)001502234.1)[SEQ ID NO: 34]:

MAAHSILDLSGLDRESQIDLQRPLKARPGKAKDLSSGSACTFRLALIQLQVSSVKSDNLTRACGLVREAAAQGAKIVCLPECFNSPYGTNYFPQYAEKIPGESTQKLSEVAKECSIYLIGGSIPEEDAGKLYNTCAVFGPDGALLVKHRKLHLFDIDVPGKITFQESKTLSPGDSFSTFDTPYCRVGLGICYDLRFAELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRGRAVDNQVYVATASPARDDKASYVAWGHSTVVTPWGEVLATAGTEEMIV YSDIDLKKLAEIR QQIPIFSQKRLDLYAVEAKKP

Xenopus (Silurana) tropicalis amino acid sequence (Genbank accessionNP_(—)001016633.1) [SEQ ID NO: 35]:

MAKFRLSLVQFLVSPVKSENLNRACKLIKEAAQKGAQIVALPECFNSPYGTKYFPEYAEKIPGESTERLSQVAKECGIYLIGGSIPEEDSGKLYNTCAVFGPDGTLLVKHRKIHLFDIDVPGKIRFQESETLSPGDSFSVFETPYCKVGVGICYDIRFAELAQLYSKKGCQLLVYPGAFNMTTGPAHWELLQRARALDNQVYVATASPARDEKASYVAWGHSTIVSPWGEVIAKAGSEETVISADIDLEYLAEIREQIPIRRQRRHDLYSVEEKKN

Danio rerio amino acid sequence (Genbank accession AAQ97821.1) [SEQ IDNO: 36]:

MSKFRLAVVQLHVSKIKADNLGRAQTLVTEAAGQGAKVVVLPECFNSPYGTGFFKEYAEKIPGESTQVLSETAKKCGIYLVGGSIPEEDGGKLYNTCSVFGPDGTLLVTHRKIHLFDIDVPGKIRFQESETLSPGKSLSMFETPYCKVGVGICYDIRFAELAQIYAKKGCQLLVYPGAFNMTTGPAHWELLQRGRAVDNQVYVATASPARDETASYVAWGHSSVINPWGEVISKAGSEESVVYADIDLQYLADVRQQIPITKQRRNDLYSVNSVQEG

Nematostella vectensis amino acid sequence (Genbank accessionXP_(—)001622809.1) [SEQ ID NO: 37]:

MAVPILVFRIGLVQLAVTANKLQNLQRAREKIKEAVAAGAKIVALPECFNSPYGTQYFKDYAEEIPGESSNMLAEVAKETGAYIVGGSIPERASNGKLYNTSLSYDPSGNLMGKHRKIHLFDIDVPGKIRFQESEVLSPGENLTILDTEYCKIGIGICYDMRFPELAQLYAKKGCHLLLYPGAFNMTTGPAHWELLTRARALDNQLYVATISPARDDNATYIAWGHSTVVNPWGKIVSKADHTEQILYAEIDLKYLNEVRSQIPVQFQKRDDVYELQVK

Mus musculus amino acid sequence (Genbank accession NP_(—)075664.1) [SEQID NO: 38]:

MSTFRLALIQLQVSSIKSDNLTRACSLVREAAKQGANIVSLPECFNSPYGTTYFPDYAEKIPGESTQKLSEVAKESSIYLIGGSIPEEDAGKLYNTCSVFGPDGSLLVKHRKIHLFDIDVPGKITFQESKTLSPGDSFSTFDTPYCKVGLGICYDMRFAELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRARAVDNQVYVATASPARDDKASYVAWGHSTVVDPWGQVLTKAGTEETILYSDIDLKKLAEIRQQIPILKQKRADLYTVESKKP

The Arabidopsis thaliana ω-amidase amino acid sequence was aligned withthe amino acid sequence of other putative plant Ω-amidases and withother putative animal Ω-amidases to identify conserved regions. Theresults of the sequence alignments are shown in FIG. 2 and FIG. 3.Regions of homology are depicted in color shading and in the consensussequences (SEQ ID NO: 44 for FIG. 2, and SEQ ID NO: 45 for FIG. 3).

Additional homologs of the Arabidopsis thaliana Ω-amidase are listed inTable 1 below.

TABLE 1 Genbank Accession No. Source Organism AAL91613.1 Arabidopsisthaliana NP_196765.2 Arabidopsis thaliana XP_002871509.1 Arabidopsislyrata subsp. lyrata NP_974769.1 Arabidopsis thaliana XP_002309478.1Populus trichocarpa XP_002279687.1 Vitis vinifera NP_001146676.1 Zeamays NP_001146295.1 Zea mays NP_001049134.1 Oryza sativa Japonica GroupXP_002516116.1 Ricinus communis XP_001766085.1 Physcomitrella patenssubsp. patens XP_001756522.1 Physcomitrella patens subsp. patensXP_002969787.1 Selaginella moellendorffii XP_002985119.1 Selaginellamoellendorffii XP_002948137.1 Volvox carteri f. nagariensisXP_001690839.1 Chlamydomonas reinhardtii NP_001057093.1 Oryza sativaJaponica Group XP_002468410.1 Sorghum bicolor NP_064587.1 Homo sapiensXP_001089575.2 Macaca mulatta XP_001502234.1 Equus caballusXP_002502298.1 Micromonas sp. RCC299 XP_526254.2 Pan troglodytesXP_535718.2 Canis familiaris XP_002716659.1 Oryctolagus cuniculusNP_001033222.1 Bos taurus NP_001029298.1 Rattus norvegicusNP_001016633.1 Xenopus (Silurana) tropicalis NP_001085409.1 Xenopuslaevis XP_002758928.1 Callithrix jacchus XP_003064056.1 Micromonaspusilla CCMP1545 NP_001135127.1 Salmo salar XP_001622809.1 Nematostellavectensis NP_991174.2 Danio rerio XP_002594716.1 Branchiostoma floridaeNP_075664.1 Mus musculus XP_001370849.1 Monodelphis domesticaNP_001090454.1 Xenopus laevis XP_002999170.1 Phytophthora infestansT30-4 XP_002917137.1 Ailuropoda melanoleuca XP_002741281.1 S accoglossuskowalevskii XP_002131764.1 Ciona intestinalis NP_594154.1Schizosaccharomyces pombe 972h- XP_001742101.1 Monosiga brevicollis MX1XP_416604.2 Gallus gallus XP_002194275.1 Taeniopygia guttataXP_001599587.1 Nasonia vitripennis XP_002410555.1 Ixodes scapularisXP_003035898.1 Schizophyllum commune H4-8 XP_002183613.1 Phaeodactylumtricornutum CCAP 1055/1 XP_001875493.1 Laccaria bicolor S238N-H82XP_002112209.1 Trichoplax adhaerens XP_636983.1 Dictyostelium discoideumAX4 XP_002158547.1 Hydra magnipapillata XP_002839272.1 Tubermelanosporum Mel28 XP_307722.3 Anopheles gambiae str. PESTXP_001819629.1 Aspergillus oryzae RIB40 Aspergillus flavus NRRL3357XP_001268376.1 Aspergillus clavatus NRRL 1 ZP_08115581.1Desulfotomaculum nigrificans DSM 574 YP_001320997.1 Alkaliphilusmetalliredigens QYMF XP_369268.1 Magnaporthe oryzae 70-15 XP_002626458.1Ajellomyces dermatitidis SLH14081 XP_751200.1 Aspergillus fumigatusAf293 XP_001657673.1 Aedes aegypti XP_002173486.1 Schizosaccharomycesjaponicus yFS275 XP_001212538.1 Aspergillus terreus NIH2624XP_001258462.1 Neosartorya fischeri NRRL 181 XP_002434512.1 Ixodesscapularis XP_960906.1 Neurospora crassa OR74A XP_002847679.1Arthroderma otae CBS 113480 XP_967861.1 Tribolium castaneumXP_002426154.1 Pediculus humanus corporis XP_003176259.1 Arthrodermagypseum CBS 118893 XP_500602.1 Yarrowia lipolytica XP_001428419.1Paramecium tetraurelia strain d4-2 XP_003014235.1 Arthroderma benhamiaeCBS 112371 XP_001393123.1 Aspergillus niger CBS 513.88 ZP_03608460.1Methanobrevibacter smithii DSM 2375 XP_002147261.1 Penicillium marneffeiATCC 18224 ZP_03293831.1 Clostridium hiranonis DSM 13275 XP_002290043.1Thalassiosira pseudonana CCMP1335 XP_003065597.1 Coccidioides posadasiiC735 delta SOWgp XP_001588734.1 Sclerotinia sclerotiorum 1980YP_001273073.1 Methanobrevibacter smithii ATCC 35061 >Methanobrevibacter smithii DSM 2374 XP_001552800.1 Botryotiniafuckeliana B05.10 XP_446414.1 Candida glabrata CBS 138 XP_002792830.1Paracoccidioides brasiliensis Pb01 XP_001998501.1 Drosophila mojavensisYP_003780301.1 Clostridium ljungdahlii DSM 13528 NP_013455.1Saccharomyces cerevisiae S288c XP_002404736.1 Ixodes scapularisYP_001086961.1 Clostridium difficile 630 ZP_05328587.1 Clostridiumdifficile QCD-63q42 ZP_05399936.1 Clostridium difficile QCD-23m63Clostridium difficile NAP08 Clostridium difficile NAP07 YP_001113615.1Desulfotomaculum reducens MI-1 XP_001247884.1 Coccidioides immitis RSXP_390426.1 Gibberella zeae PH-1 XP_003025334.1 Trichophyton verrucosumHKI 0517 XP_002052999.1 Drosophila virilis ZP_07325748.1 Acetivibriocellulolyticus CD2 ZP_05349666.1 Clostridium difficile ATCC 43255

Accordingly, in certain embodiments, the Ω-amidase transgene encodes apolypeptide having an amino acid sequence that is at least 75%, at least80%, at least 85%, at last 90%, at last 91%, at last 92%, at last 93%,at last 94%, at last 95%, at last 96%, at last 97%, at last 98%, at last99%, or 100% identical to an amino acid sequence encoded by apolypeptide selected from AAL91613.1, ACN30911.1, ABK22312.1,ACJ85250.1, AAQ97821.1, CBJ25483.1, EFN54567.1, NP_(—)196765.2,XP_(—)002871509.1, NP_(—)974769.1, XP_(—)002309478.1, XP_(—)002279687.1,NP_(—)001146676.1, NP_(—)001146295.1, NP_(—)001049134.1,XP_(—)002516116.1,

XP_(—)001766085.1, XP_(—)001756522.1, XP_(—)002969787.1,XP_(—)002985119.1, XP_(—)002948137.1, XP_(—)001690839.1,NP_(—)001057093.1, XP_(—)002468410.1, NP_(—)064587.1, XP_(—)001089575.2,XP_(—)001502234.1, XP_(—)002502298.1, XP_(—)526254.2, XP_(—)535718.2,XP_(—)002716659.1, NP_(—)001033222.1, NP_(—)001029298.1,NP_(—)001016633.1, NP_(—)001085409.1, XP_(—)002758928.1,XP_(—)003064056.1, NP_(—)001135127.1, XP_(—)001622809.1, NP_(—)991174.2,XP_(—)002594716.1,

NP_(—)075664.1, XP_(—)001370849.1, NP_(—)001090454.1, XP_(—)002999170.1,XP_(—)002917137.1, XP_(—)002741281.1, XP_(—)002131764.1, NP_(—)594154.1,XP_(—)001742101.1, XP_(—)416604.2, XP_(—)002194275.1, XP_(—)001599587.1,XP_(—)002410555.1, XP_(—)003035898.1, XP_(—)002183613.1,XP_(—)001875493.1, XP_(—)002112209.1, XP_(—)636983.1, XP_(—)002158547.1,XP_(—)002839272.1, XP_(—)307722.3, XP_(—)001819629.1, XP_(—)001268376.1,ZP_(—)08115581.1, YP_(—)001320997.1, XP_(—)369268.1, XP_(—)002626458.1,XP_(—)751200.1, XP_(—)001657673.1, XP_(—)002173486.1, XP_(—)001212538.1,XP_(—)001258462.1, XP_(—)002434512.1, XP_(—)960906.1, XP_(—)002847679.1,XP_(—)967861.1, XP_(—)002426154.1, XP_(—)003176259.1, XP_(—)500602.1,XP_(—)001428419.1, XP_(—)003014235.1, XP_(—)001393123.1,ZP_(—)03608460.1, XP_(—)002147261.1, ZP_(—)03293831.1,XP_(—)002290043.1, XP_(—)003065597.1, XP_(—)001588734.1,YP_(—)001273073.1, XP_(—)001552800.1,

XP_(—)446414.1, XP_(—)002792830.1, XP_(—)001998501.1, YP_(—)003780301.1,NP_(—)013455.1, XP_(—)002404736.1, YP_(—)001086961.1, ZP_(—)05328587.1,ZP_(—)05399936.1, YP_(—)001113615.1, XP_(—)001247884.1, XP_(—)390426.1,XP_(—)003025334.1, XP_(—)002052999.1, YP_(—)769862.1, ZP_(—)07325748.1,ZP_(—)05349666.1, YP_(—)471237.1, YP_(—)002977603.1, YP_(—)001202760.1,ZP_(—)07592670.1, and NP_(—)386723.1. In other embodiments, theΩ-amidase transgene is incorporated into the genome of the transgenicplants.

Certain aspects of the present disclosure relate to an Ω-amidasetransgene that is operably linked to a root-preferred promoter. As usedherein, a “root-preferred promoter” refers to expression driven by apromoter that is selectively enhanced in root cells or tissues, incomparison to one or more non-root cells or tissues. For example, aroot-preferred promoter may preferentially drive high levels ofexpression of a gene in root cells or tissue but may still drive lowlevels of expression of the gene in other non-root cells or tissues,such as leaves. Root tissues include but are not limited to at least oneof root cap, apical meristem, protoderm, ground meristem, procambium,endodermis, cortex, vascular cortex, epidermis, and the like.

In certain other embodiments, 2-oxoglutaramate levels in root tissuesare decreased in order to increase the leaf-to-root ratio thereof byincreasing the natural breakdown of 2-oxoglutaramate in root tissue. Forexample, the breakdown of 2-oxoglutaramate in root tissue may beincreased by upregulating Ω-amidase activity in the roots. Thus, in oneembodiment, an Ω-amidase transgene, including without limitation any ofthe Ω-amidase genes and coding sequences disclosed herein, is introducedinto the plant under the control of a root-preferred promoter, such asthe rolD promoter of Agrobacterium rhizogenes (Kamo and Bowers, 1999,Plant Cell Reports 18: 809-815 and references cited therein). The rolDpromoter controls the expression of rolD, which functions to promoteroot elongation. GUS protein expression under control of therolD-promoter has been shown to yield mainly root-preferred GUSexpression (Leach and Aoyagi, 1991, Plant Sci. 79, 69-76).Root-preferred promoters may be either constitutive or inducible.

Additional constitutive and/or inducible root-preferred promotersinclude, without limitation, the RolD-2 promoter; glycine rich promoters(GRP); ADH promoters, including the maize ADH1 promoter (Kyozuka, J etal., 1994, The Plant Cell 6:799-810); PHT promoters, including the Pht1gene family promoters (Schunmann et al., 2004, J. Experimental Botany55:855-865); metal uptake protein promoters, including the maizemetallothionein promoter (Diehn, S, 2006 Maize, U.S. Pat. ApplicationPublication US 2006/0005275); the 35S CaMV domain A promoter (Elmayan, Tand M. Tepfer. 1995, Transgenic Research 4:388-396); the pDJ3S, SIREO,and pMe1 promoters (Arango et al., Plant Cell Rep. 2010 June; 29(6):651-9. Epub 2010 Apr. 6.); the Sad1 and Sad2 promoters (U.S. PatentApplication Publication US 2008/0244791); the TobRB7 promoter (Yamamotoet al., 1991, Plant Cell 3:371-3); the RCc3 promoter (Xu et al., 1995,Plant Mol. Biol. 27:237-248); the FaRB7 promoter (Vaugn et al., Exp.Bot. (2006) 57 (14): 3901-3910); the SPmads promoter (Noh et al.,American Society of Plant Biologists, Plant Biology 2005 Conference,Abstract #1097); the IDS2 promoter (Kobayashi et al., The Plant Journal,Vol. 36(6): 780-793, December 2003); the pyk10 promoter (Nitz et al.,Plant Sci. 2001 July; 161(2):337-346); the Pt2L4 promoter (De Souza etal., Genet. Mol. Res. 8 (1): 334-344 (2009)); the Lbc3 leghemoglobinpromoter (Bak, K, et al., 1993, The Plant Journal 4(3) 577-580); thePEPC promoter (Kawamura et al. 1990, J. Biochem 107: 165-168); the Gns1Glucanase root promoter (Simmons, C et al., 1992, Plant MolecularBiology 18: 33-45), the 35S.sup.2promoter (Elmayan, T and M. Tepfer.1995, Transgenic Research 4:388-396); the GI4 and GI5 promoters(European Patent EP 1 862 473 B1; and the GRP promoter. Additionally,any of the disclosed root-preferred promoters may be in a truncated formthat contains only the core domain or functional domain of the promotersufficient to drive expression in root tissue. Moreover, root-preferredpromoters also include isoforms of any of the root-preferred promotersdisclosed herein. For example, the RolD-2 promoter is one of severaltruncated isoforms of the RolD promoter described in Leach and Aoyagi,1991, Plant Sci. 79, 69-76. Moreover, Leach and Aoyagi describe theRolD2 promoter as being a highly root-preferred promoter. Accordingly, aroot-preferred promoter also includes any of the RolD isomers describedby Leach and Aoyagi.

Thus, in certain embodiments, the root-preferred promoter is selectedfrom RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRPpromoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 genefamily promoter, metal uptake protein promoter, maize metallothioneinprotein promoter, 35S CaMV domain A promoter. pDJ3S promoter, SIREOpromoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter,RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanaseroot promoter, 35S.sup.2promoter, GI4 promoter, GI5 promoter, and GRPpromoter.

In stable transformation embodiments, one or more copies of theΩ-amidase transgene become integrated into the genome of the transgenicplant, thereby providing increased Ω-amidase enzyme capacity into theroot tissue of the plant, which serves to mediate synthesis of2-oxoglutaramate, which in turn signals metabolic gene expression,resulting in increased nitrogen use efficiency, which in turn results inincreased plant growth and the enhancement of other agronomiccharacteristics.

In other embodiments, root-preferred expression of the Ω-amidasetransgene results in an increased leaf-to-root ratio of 2-oxoglutaramterelative to a plant of the same species that does not contain anΩ-amidase transgene. In certain preferred embodiments, the leaf-to-rootratio of 2-oxoglutaramate is at least two times, at least three times,at least four times, at least five times, at least six times, at leastseven times, at least eight times, at least nine times, at least tentimes, or more higher than that of a plant of the same species that doesnot contain an Ω-amidase transgene.

In further embodiments, the transgenic plant has increased nitrogen useefficiency. The disclosed transgenic plants with increased nitrogen useefficiency may further contain other transgenes known to increasenitrogen utilization efficiency, including without limitation thosedescribed in U.S. Pat. No. 7,560,626.

Impairing the Breakdown of 2-Oxoglutaramate in Leaf Tissues byInhibiting Ω-Amidase Activity:

Other aspects of the present disclosure relate to transgenic plantshaving inhibited expression of endogenous Ω-amidase in leaf tissue.

Accordingly, in certain embodiments, the breakdown of 2-oxoglutaramateor its analogs may be impaired by decreasing Ω-amidase activity in orderallow accumulation of 2-oxoglutaramate in the leaves, thereby increasingthe leaf-to-root ratio. More specifically, the following approaches toimpeding the 2-oxoglutaramate breakdown pathway may be used.

In one specific embodiment, the normal metabolic breakdown of2-oxogluataramate catalyzed by an Ω-amidase enzyme, including but notlimited to any of the Ω-amidase enzymes disclosed herein, is inhibitedin leaf tissue of any of the disclosed transgenic plants by theapplication of a chemical inhibitor, including without limitation6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropylfluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate,silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.Accordingly, in certain embodiments, endogenous Ω-amidase expression inleaf tissue of any of the disclosed transgenic plants is inhibited by achemical inhibitor selected from 6-diazo-5-oxo-nor-leucine,p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide,phenylmercuriacetate, Iodoacetate, silver nitrate,chloromercuricphenylsulfonic acid, and copper sulfate.

In another embodiment, Ω-amidase function may be inhibited in leaftissue of any of the disclosed transgenic plants by genetically impedingthe transcription and/or translation of an Ω-amidase gene, including butnot limited to the Ω-amidase genes and coding sequences disclosedherein. Methods for impeding Ω-amidase expression and function include,without limitation, recessive gene disruption and dominant genesilencing.

As used herein, “recessive gene disruption” refers to mutating a targetΩ-amidase sequence to eliminate either expression or function. Methodsfor mutating a target sequence are known in the art, and include,without limitation, the generation of mutations via chemical orradiation damage followed by isolation of the mutant. In addition, knownmolecular biology approaches for decreasing the expression of afunctional phenotype may be used, and include without limitation,various knockout or knockdown methods. These methods capitalize uponknowledge of sequence either in the gene of interest or in the DNAsequence flanking the gene. Such sequences are then examined to findsuitable sequences that can be targeted to accomplish either excision ofthe target gene or fragments of the gene. Thus, in certain embodiments,the endogenous Ω-amidase expression in leaf tissue of any of thedisclosed transgenic plants is inhibited by a recessive gene disruptionselected from a mutant Ω-amidase gene that eliminates endogenousΩ-amidase expression, an endogenous Ω-amidase knockout mutant, and anendogenous Ω-amidase knockdown mutant.

As used herein, “dominant gene silencing” refers to inducing ordestroying/inhibiting the mRNA transcript of the gene, a means whichprovides the benefit of being done in a spatial or temporal manner bythe selection of specific promoters. Of the dominant gene silencingapproaches, dsRNA-triggered RNAi is one of the most powerful and themost efficient at gene silencing, and allows one to enhance orcapitalize upon a natural regulatory mechanism which destroys intactmRNA by providing an antisense oligonucleotide that is specific for anendogenous Ω-amidase gene (For review, see, Behlke, 2006, MolecularTherapy 13(4): 644-670; see also, Tang and Galili, 2004, TrendsBiotechnology 22:463-469; Rajewsky and Socci, 2004, DevelopmentalBiology 267:529-535; Hamilton et al., 2002, EMBO J. 21:4671-46794 In oneembodiment, a construct comprising a suitable RNAi sequence under thecontrol of a leaf specific promoter such as the RuBisCo small subunitpromoter is introduced into the plant in order to silence Ω-amidaseprotein expression. Accordingly, in certain embodiments, the endogenousΩ-amidase expression in leaf tissue of any of the disclosed transgenicplants is inhibited by an RNAi antisense oligonucleotide that isspecific for an endogenous Ω-amidase gene.

In certain embodiments, inhibition of endogenous Ω-amidase expression inleaf tissue results in an increased leaf-to-root ratio of2-oxoglutaramte relative to a plant of the same species that does notcomprise inhibited endogenous Ω-amidase expression in leaf tissue. Incertain preferred embodiments, the leaf-to-root ratio of2-oxoglutaramate is at least two times, at least three times, at leastfour times, at least five times, at least six times, at least seventimes, at least eight times, at least nine times, at least ten times, ormore higher than that of a plant of the same species that does notcomprise inhibited endogenous Ω-amidase expression in leaf tissue. Infurther embodiments, the transgenic plant has increased nitrogen useefficiency.

Similarly, the expression of the substrate GS and/or the catalyticprotein GPT may be impaired in root tissue using any of the approachesdisclosed herein.

Embodiments Relating to Transgenic Plants with Increased Ω-AmidaseActivity in Root Tissues and Inhibited Ω-Amidase Activity in LeafTissue:

Certain aspects of the present disclosure relate to transgenic plantswith increased Ω-amidase expression in root tissue and inhibitedendogenous Ω-amidase expression in leaf tissue, which results in anincreased leaf-to-root ratio of 2-oxoglutaramte.

Accordingly, in certain embodiments, transgenic plants containing anΩ-amidase transgene that is operably linked to a root-preferredpromoter, further have inhibited endogenous Ω-amidase expression in leaftissue. Exemplary transgenic plants Ω-amidase transgenes, androot-preferred promoters are as described in previous sections. In otherembodiments, the endogenous Ω-amidase expression in leaf tissue isinhibited by recessive gene disruption, dominant gene silencing, or achemical inhibitor. In still other embodiments, the endogenous Ω-amidaseexpression in leaf tissue is inhibited by a recessive gene disruptionselected a mutant Ω-amidase gene that eliminates endogenous Ω-amidaseexpression, an endogenous Ω-amidase knockout mutant, and an endogenousΩ-amidase knockdown mutant. In yet other embodiments, the endogenousΩ-amidase expression in leaf tissue is inhibited by an RNAi antisenseoligonucleotide that is specific for an endogenous Ω-amidase gene. Infurther embodiments, the endogenous Ω-amidase expression in leaf tissueis inhibited by a chemical inhibitor selected from6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropylfluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate,silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.In further embodiments, the transgenic plant has an increasedleaf-to-root ratio of 2-oxoglutaramte. In certain preferred embodiments,the leaf-to-root ratio of 2-oxoglutaramate is at least two times, atleast three times, at least four times, at least five times, at leastsix times, at least seven times, at least eight times, at least ninetimes, at least ten times, or more higher than that of an unmodifiedplant of the same species. In still further embodiments, the transgenicplant has increased nitrogen use efficiency.

In certain embodiments, transgenic plants having inhibited expression ofendogenous Ω-amidase in leaf tissue relative to a plant of the samespecies that does not comprise inhibited expression of endogenousΩ-amidase in leaf tissue, where the endogenous Ω-amidase expression inleaf tissue is inhibited by recessive gene disruption or dominant genesilencing of at least one endogenous Ω-amidase gene, further contain anΩ-amidase transgene, where the Ω-amidase transgene is operably linked toa root-preferred promoter. Exemplary transgenic plants, recessive genedisruption, dominant gene silencing, Ω-amidase transgenes, androot-preferred promoters are as described in previous sections. Infurther embodiments, the transgenic plant has an increased leaf-to-rootratio of 2-oxoglutaramte. In certain preferred embodiments, theleaf-to-root ratio of 2-oxoglutaramate is at least two times, at leastthree times, at least four times, at least five times, at least sixtimes, at least seven times, at least eight times, at least nine times,at least ten times, or more higher than that of an unmodified plant ofthe same species. In still further embodiments, the transgenic plant hasincreased nitrogen use efficiency.

Expression of Glutamine Phenylpyruvate Transaminase and GlutamineSynthetase:

Other aspects of the present disclosure relate to transgenic plants withincreased Ω-amidase expression in root tissue or inhibited endogenousΩ-amidase expression in leaf tissue that further contain increasedexpression of glutamine phenylpyruvate transaminase (GPT) and/orglutamine synthetase (GS).

In a particular embodiment, any of the transgenic plants disclosedherein further over-express the GPT protein, which is directly involvedin the synthesis of 2-oxoglutaramate, which results in higherleaf-to-root ratios of the 2-oxoglutaramate compound. In a relatedembodiment, any of the transgenic plants disclosed herein furtherover-express the GPT protein and the GS protein. These transgenic plantsfurther containing GPT and GS have an even higher leaf-to-root ratios of2-oxoglutaramate, resulting in a further increase in nitrogen useefficiency. This increase in nitrogen use efficiency also results inplants that grow faster, produce greater seed and fruit/pod yields,display earlier and more productive flowering, demonstrate increasedtolerance to high salt conditions, and produce superior biomass yields(See, co-owned, co-pending U.S. patent application Ser. No. 12/551,271).

More particularly, applicants have determined that the over-expressionof GPT and GS in transgenic plants results in a disproportionateincrease in the relative concentrations of 2-oxoglutaramate in thefoliar and below ground tissues. Moreover, the ratio of theconcentration of 2-oxoglutaramate in above ground tissue to below groundtissue (leaf-to-root ratio) is positively correlated with plant biomass.Faster growing, larger genetically-engineered plants have a greaterratio of the concentrations of 2-oxoglutaramate in leaf tissue versusroot tissue when compared to wild type plants. In one particularembodiment, transgenic tobacco plants carrying GPT and GS1 transgenesunder the control of robust constitutive promoters showed substantiallygreater leaf-to-root ratios of 2-oxoglutaramate and demonstrated highgrowth phenotypes when compared to wild type tobacco plants. Inparticular, two transgenic tobacco lines over-expressing GPT and GS1transgenes were found to have: (1) well over two-times the fresh weightof wild type plants, (2) two-times the 2-oxoglutaramate foliarconcentration compared to the wild type plants, and (3) between two- andthree-times the leaf-to-root ratio seen in wild type plants.

In a wild type or engineered plant, the ratio can be expected to reflectthe relative concentrations of 2-oxoglutaramate, as well as glutamine,the substrate from which it is made, in the leaves versus the roots ofthe plant. The actual ratios would be expected to differ from species tospecies. This is due to the fact that leaves and roots house differingfractions of the nitrogen assimilation machinery and activity, as afunction of plant species (Pate, 1980, Ann. Rev. Plant Physiol. 31:313-340). In fact, some plant species assimilate most of their nitrogenin their roots, and thus have high amino acid concentrations in theirroots and xylem sap, and lower concentrations in their leaves. Otherplants assimilate most of their nitrogen in their leaves, and thus havehigh amino acid concentrations in their leaves, and lower concentrationsin their roots. Plant species distribute themselves along a continuum ofthis distribution of labor and amino acid concentrations between leavesand roots.

In addition to the over-expression of natural GPT proteins in transgenicplant systems, genetically-engineered, enhanced GPT enzymes may bedeveloped and used to improve 2-oxoglutaramate synthesis kinetics,thereby increasing the rate of 2-oxoglutaramate accumulation in leaves.The GPT enzyme may be broadly classed as being a member of aspartateamino transferase type enzymes, based on sequence homology with knownwell characterized aspartate amino transferase enzymes. The major genesequence databases include this classification of the transferaseenzymes as a part of their sequence analysis (Gen Bank for example).Characteristically these are vitamin B6-dependent enzymes which catalyzetransamination reactions between an amino acid and a ketoacid. Thekinetic properties of these many (1000) transaminases differ in suchproperties as substrate specificities, binding constants, maximalvelocity (Vmax) and unimolecular turnover rates (Kcat). The specificarginine residues involved directly in the hydrogen-bonding of thesubstrate dicarboxlyic acid substrates have been highly conserved(Fotheringham et al., 1986, Biochem J. 234:593-604; Seville et al.,1988, Biochemistry 27:8344-8349: Jager et al., 1992, FEBS Lett.306:234-238) and thus the changes in specificities and kineticproperties are often conferred by changes in other amino acid residues.The enzyme's performance has proven to be very sensitive to subtlechanges in the structure of the residue, for example the addition of asingle CH₂ group in a residue not in direct contact with eithersubstrate or co-factor (Jansonius and Vincent, 1987; Seville et al.,1988, supra). Various studies have shown that it is possible to changean aspartate amino transferase enzyme's properties with directedmutation of the wild type protein (Kohler et al., 1994, Biochemistry33:90-97; Jager et al., 1994, Protein Engineering. 7:605-612).

Within the plant GPT sequences, the region NLGQGFP (SEQ ID NO: 18) ishighly conserved (and completely conserved among soybean, grape, ricehordeum, and Arabidopsis sequences (See, U.S. patent application Ser.No. 12/551,271 and U.S. patent application Ser. No. 12/551,193).Applicants have used such a directed mutation approach to generate amutant GPT from the natural Arabidopsis GPT, by substituting V (valine)for the F (phenylalanine) residue in the wild type sequence. Theresulting GPT/F:V mutant was expressed in E. coli, using the common PETvector system, and showed improved maximal velocity and unimolecularturnover. Maximal velocity was determined using the formula:Vmax=Kcat[E] tot. The apparent unimolecular rate constant Kcat is alsocalled turnover number and denotes the maximum number of enzymaticreactions catalyzed per second.

Vmax for the mutant increased to 6.04, a 20% increase over the wild typeVmax value of 5.07. Care was taken to assure that the same amount ofprotein was used in these experiments and thus the relationship ofVmax=Kcat[E]tot can be applied to show that the mutant's unimolecularturnover rate has increased. The glutamine Km for the mutant is 0.75millimolar, a slight increase from the 0.30 mM Km measured for the wildtype enzyme. This mutant GPT enzyme may be expected to produce moreproduct, the 2-oxoglutaramate, per unit time than the wild type GPT whenthe concentration of the substrate glutamine is present in millimolar orgreater quantities, thus assuring that the mutant is saturating. Asurvey of the plant and agricultural literature shows that wellnourished plants contain millimolar glutamine concentrations (Dzuibanyet al., 1998, Plants 206:515-522; Knight and Weissman, 1982, PlantPhysiol 70:1683-1688; Sivasankar and Oaks, 1995, Plant Physiol. 107:1225-1231; Yanagisawa et al., 2004, Proc. Natl. Acad. Sci. USA101:7833-7838; Udy and Dennison, 1997, Australia J Experimental MarineBiology and Ecology 217:253-277).

The amino acid sequence of the GPT/F:V mutant protein is as follows (Vsubstitution shown in bold) [SEQ ID NO: 1]:

MYLDINGVMIKQFSFKASLLPFSSNFRQSSAKIHRPIGATMTTVSTQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQGVPNFDGPDFVKEAAIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAMLGLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHNPTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCEYLIEEVGVVAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLK R KV

Two other substitution mutants were made at this residue, and one wasalso expressed in E. coli and analyzed kinetically (F:L mutation), butshowed a higher (less desirable) Km value of 1.98 mM and a decreasedVmax value of 4.0.

In another approach to this aspect of the present disclosure, GPT and/orGS transgenes may be designed to utilize the codon usage preferred bythe target plant species. Codon usage in plants is well established tovary particularly between monocots and dicots; in general, monocots seemto have higher GC usage overall with a very pronounced GC preference atthe third base position (Kawabe and Miyashita, 2003, Genes & GeneticSystems 78(5): 343-52). Codon usage bias has been correlated withprotein expression levels (Hiroaka et al., 2009). Thus one skilled inthe art can refer to such sources as the Codon Usage Database or thework of Kawabe and Miyashita comparing several monocots and dicots orother genome sequence information and use or deduce the preferred codonusage for the target plant and simply design and synthesize theoptimized gene sequence.

In yet a further approach to this aspect of the present disclosure,consensus engineering of the GPT and/or GS structures is used togenerate consensus variants showing significant increases in proteinstability in order to improve the amount of GPT activity in the plant.In this approach the native sequence is modified to more closelyresemble a consensus sequence derived from the alignment of numerousproteins of a particular family (Schiller et al., 1994, J Mol. Biol.240:188-192).

Accordingly, in certain embodiments, any of the transgenic plants withmodulated Ω-amidase expression disclosed herein further contain a GPTtransgene. In certain embodiments, the GPT transgene is a GPT/F:V mutantgiven by SEQ ID NO:1. In other embodiments, any of the transgenic plantsdisclosed herein further contain a GPT transgene and a GS transgene. Inyet other embodiments, the GPT transgene and GS transgene are eachoperably linked to a leaf-preferred promoter. As used herein, a“leaf-preferred promoter” refers to expression driven by a promoter thatis selectively enhanced in leaf cells or tissues, in comparison to oneor more non-leaf cells or tissues. For example, a leaf-preferredpromoter may preferentially drive high levels of expression of a gene inleaf cells or tissue but may also drive low levels of expression of thegene in other non-leaf cells or tissues, such as roots.

In other embodiments, any of the disclosed transgenes are codonoptimized for expression in the plant. In further embodiments, thetransgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramte.In certain preferred embodiments, the leaf-to-root ratio of2-oxoglutaramate is at least two times, at least three times, at leastfour times, at least five times, at least six times, at least seventimes, at least eight times, at least nine times, at least ten times, ormore higher than that of an unmodified plant of the same species. Instill further embodiments, the transgenic plant has increased nitrogenuse efficiency.

Increasing 2-Oxoglutaramate Biosynthesis in Leaf Tissues by GeneActivation:

As yet another approach, genes encoding proteins involved in themetabolic pathway which produces 2-oxoglutaramate, which genes may be“silent” and not expressed in particular cell type in a plant, may beactivated, or turned-on, via gene activation methodologies, such as thehomologous recombination methods developed by Transkaryotic Therapies,Inc. (see, for example U.S. Pat. No. 6,187,305). In these methods, theendogenous regulatory region of a gene is replaced with a regulatorysequence from a different gene or a novel regulatory whose presence inthe cell results in expression of the gene. Such regulatory sequencesmay be comprised of promoters, enhancers, scaffold-attachment regions,negative regulatory elements, transcriptional initiation sites,regulatory protein binding sites or combinations of these sequences. Asa result, an endogenous copy of a gene encoding a desired gene productis turned on and expressed, and an exogenous copy of the gene need notbe introduced.

In a related embodiment, transcription factor upregulation orover-expression may be used to increase the transcription of genes whichpromote higher leaf-to-root ratios of 2-oxoglutaramate and/or itsanalogs. In one embodiment, the Dof-1 transcription factor is introducedas a transgene in order to induce the up-regulation of genes encodingenzymes for carbon skeleton production, a marked increase in amino acidcontent and a reduction in the glucose level, as previously reported intransgenic Arabidopsis. Over-expression of the Dof-1 transcriptionfactor has been shown to improved nitrogen assimilation and growth underlow-nitrogen conditions (Yanagisawa et al., 2004, PNAS 101:7833-7838).In this report, the transcription factor was expressed constitutively inthe plant. Over expression of the Dof-1 transcription factor, alone, orin combination with other measures such as the over-expression of GS andGPT (or functionally-improved mutants thereof) in above-ground planttissues can be expected to increase the leaf to root ratio of2-oxoglutaramate.

Accordingly, in certain embodiments, any of the transgenic plants withmodulated Ω-amidase expression disclosed herein also contain increasedendogenous GPT expression, where the endogenous GPT expression isincreased by gene activation. In still further embodiments, any of thetransgenic plants disclosed herein contain increased endogenous GSexpression, where the endogenous GS expression is increased by geneactivation. In further embodiments, the transgenic plant has anincreased leaf-to-root ratio of 2-oxoglutaramte. In certain preferredembodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least twotimes, at least three times, at least four times, at least five times,at least six times, at least seven times, at least eight times, at leastnine times, at least ten times, or more higher than that of anunmodified plant of the same species. In still further embodiments, thetransgenic plant has increased nitrogen use efficiency.

Suitable Transgenic Plants:

Certain aspects of the present disclosure relate to transgenic plants.The transgenic plants disclosed herein may be any vascular plant of thephylum Tracheophyta, including angiosperms and gymnosperms. Angiospermsmay be a monocotyledonous (monocot) or a dicotyledonous (dicot) plant.Important monocots include those of the grass families, such as thefamily Poaceae and Gramineae, including plants of the genus Avena (Avenasativa, oats), genus Hordeum (i.e., Hordeum vulgare, Barley), genusOryza (i.e., Oryza sativa, rice, cultivated rice varieties), genusPanicum (Panicum spp., Panicum virgatum, Switchgrass), genus Phleum(Phleum pratense, Timothy-grass), genus Saccharum (i.e., Saccharumofficinarum, Saccharum spontaneum, hybrids thereof, Sugarcane), genusSecale (i.e., Secale cereale, Rye), genus Sorghum (Sorghum vulgare,Sorghum), genus Triticum (wheat, various classes, including T. aestivumand T. durum), genus Fagopyrum (buckwheat, including F. esculentum),genus Triticosecale (Triticale, various hybrids of wheat and rye), genusChenopodium (quinoa, including C. quinoa), genus Zea (i.e., Zea mays,numerous varieties) as well as millets (i.e., Pennisetum glaucum)including the genus Digitaria (D. exilis).

Important dicots include those of the family Solanaceae, such as plantsof the genus Lycopersicon (Lycopersicon esculentum, tomato), genusCapiscum (Capsicum annuum, peppers), genus Solanum (Solanum tuberosum,potato, S. lycopersicum, tomato); genus Manihot (cassava, M. esculenta),genus Ipomoea (sweet potato, I. batatas), genus Olea (olives, includingO. europaea); plants of the Gossypium family (i.e., Gossypium spp., G.hirsutum, G. herbaceum, cotton); the Legumes (family Fabaceae), such aspeas (Pisum spp, P. sativum), beans (Glycine spp., Glycine max(soybean);Phaseolus vulgaris, common beans, Vigna radiata, mung bean), chickpeas(Cicer arietinum)), lentils (Lens culinaris), peanuts (Arachishypogaea); coconuts (Cocos nucifera) as well as various other importantcrops such as camelina (Camelina sativa, family Brassicaceae), citrus(Citrus spp, family Rutaceae), coffee (Coffea spp, family Rubiaceae),melon (Cucumis spp, family Cucurbitaceae), squash (Cucurbita spp, familyCucurbitaceae), roses (Rosa spp, family Rosaceae), sunflower (Helianthusannuus, family Asteraceae), sugar beets (Beta spp, familyAmaranthaceae), including sugarbeet, B. vulgaris), genus Daucus(carrots, including D. carota), genus Pastinaca (parsnip, including P.sativa), genus Raphanus (radish, including R. sativus), genus Dioscorea(yams, including D. rotundata and D. cayenensis), genus Armoracia(horseradish, including A. rusticana), genus Elaeis (Oil palm, includingE. guineensis), genus Linum (flax, including L. usitatissimum), genusCarthamus (safflower, including C. tinctorius L.), genus Sesamum(sesame, including S. indicum), genus Vitis (grape, including Vitisvinifera), and plants of the genus Brassica (family Brassicaceae, i.e.,broccoli, brussel sprouts, cabbage, swede, turnip, rapeseed B. napus,and cauliflower).

Other specific plants which may be transformed to generate thetransgenic plants of the present disclosure include various other fruitsand vegetables, such as apples, asparagus, avocado, banana, blackberry,blueberry, brussel sprout, cabbage, cotton, canola, carrots, radish,cucumbers, cherries, cranberries, cantaloupes, eggplant, grapefruit,lemons, limes, nectarines, oranges, peaches, pineapples, pears, plums,tangelos, tangerines, papaya, mango, strawberry, raspberry, lettuce,onion, grape, kiwi fruit, okra, parsnips, pumpkins, and spinach. Inaddition various flowering plants, trees and ornamental plants may beused to generate transgenic varietals, including without limitationlily, carnation, chrysanthemum, petunia, geranium, violet, gladioli,lupine, orchid and lilac.

In certain embodiments, the transgenic plant is selected from wheat,oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses,fruits, vegetables, flowering plants, and trees.

Other aspects of the present disclosure also relate to a progeny of anygeneration of any of the transgenic plants disclosed herein. A furtheraspect relates to a seed of any generation of the transgenic plantsdisclosed herein.

Production of Transgenic Plants:

Certain aspects of the present disclosure relate to methods forgenerating transgenic plants with increased nitrogen use efficiency.Exemplary methods for the production of transgenic plants are describedbelow. Further examples are described in co-owned, co-pending U.S.patent application Ser. Nos. 12/551,271, and 12/660,501, both of whichare incorporated in their entireties by reference herein.

Transgene Constructs/Expression Vectors:

In order to generate the transgenic plants of the present disclosure,the gene coding sequence for the desired transgene(s) must beincorporated into a nucleic acid construct (also interchangeablyreferred to herein as a/an (transgene) expression vector, expressioncassette, expression construct or expressible genetic construct), whichcan direct the expression of the transgene sequence in transformed plantcells. Such nucleic acid constructs carrying the transgene(s) ofinterest may be introduced into a plant cell or cells using a number ofmethods known in the art, including but not limited to electroporation,DNA bombardment or biolistic approaches, microinjection, and via the useof various DNA-based vectors such as Agrobacterium tumefaciens andAgrobacterium rhizogenes vectors. Once introduced into the transformedplant cell, the nucleic acid construct may direct the expression of theincorporated transgene(s) (i.e., Ω-amidase), either in a transient orstable fashion. Stable expression is preferred, and is achieved byutilizing plant transformation vectors which are able to direct thechromosomal integration of the transgene construct. Once a plant cellhas been successfully transformed, it may be cultivated to regenerate atransgenic plant.

A large number of expression vectors suitable for driving theconstitutive or induced expression of inserted genes in transformedplants are known. In addition, various transient expression vectors andsystems are known. To a large extent, appropriate expression vectors areselected for use in a particular method of gene transformation (see,infra). Broadly speaking, a typical plant expression vector forgenerating transgenic plants will comprise the transgene of interestunder the expression regulatory control of a promoter, a selectablemarker for assisting in the selection of transformants, and atranscriptional terminator sequence.

More specifically, the basic elements of a nucleic acid construct foruse in generating the transgenic plants of the present disclosure are: asuitable promoter, such as a root-preferred promoter, capable ofdirecting the functional expression of the transgene(s) in a transformedplant cell, the transgene (s) (i.e., Ω-amidase coding sequence) operablylinked to the promoter, preferably a suitable transcription terminationsequence (i.e., nopaline synthetic enzyme gene terminator) operablylinked to the transgene, and sometimes other elements useful forcontrolling the expression of the transgene, as well as one or moreselectable marker genes suitable for selecting the desired transgenicproduct (i.e., antibiotic resistance genes).

As Agrobacterium tumefaciens is the primary transformation system usedto generate transgenic plants, there are numerous vectors designed forAgrobacterium transformation. For stable transformation, Agrobacteriumsystems utilize “binary” vectors that permit plasmid manipulation inboth E. coli and Agrobacterium, and typically contain one or moreselectable markers to recover transformed plants (Hellens et al., 2000,Technical focus: A guide to Agrobacterium binary Ti vectors. TrendsPlant Sci 5:446-451). Binary vectors for use in Agrobacteriumtransformation systems typically comprise the borders of T-DNA, multiplecloning sites, replication functions for Escherichia coli and A.tumefaciens, and selectable marker and reporter genes.

So-called “super-binary” vectors provide higher transformationefficiencies, and generally comprise additional virulence genes from aTi (Komari et al., 2006, Methods Mol. Biol. 343: 15-41). Super binaryvectors are typically used in plants which exhibit lower transformationefficiencies, such as cereals. Such additional virulence genes includewithout limitation virB, virE, and virG (Vain et al., 2004, The effectof additional virulence genes on transformation efficiency, transgeneintegration and expression in rice plants using the pGreen/pSoup dualbinary vector system. Transgenic Res. 13: 593-603; Srivatanakul et al.,2000, Additional virulence genes influence transgene expression:transgene copy number, integration pattern and expression. J. PlantPhysiol. 157, 685-690; Park et al., 2000, Shorter T-DNA or additionalvirulence genes improve Agrobacterium-mediated transformation. Theor.Appl. Genet. 101, 1015-1020; Jin et al., 1987, Genes responsible for thesupervirulence phenotype of Agrobacterium tumefaciens A281. J.Bacteriol. 169: 4417-4425).

Plant Promoters:

In order to generate the transgenic plants of the present disclosure,the gene coding sequence for the desired transgene(s) is are operablylinked to a promoter in order to drive expression of the transgene. Alarge number of promoters which are functional in plants are known inthe art. In constructing Ω-amidase, GPT, or GS transgene constructs andΩ-amidase RNAi constructs, the selected promoter(s) may be constitutive,non-specific promoters such as the Cauliflower Mosaic Virus 35Sribosomal promoter (CaMV 35S promoter), which is widely employed for theexpression of transgenes in plants. Examples of other strongconstitutive promoters include without limitation the rice actin 1promoter, the CaMV 19S promoter, the Ti plasmid nopaline synthasepromoter, the alcohol dehydrogenase promoter and the sucrose synthasepromoter.

Alternatively, in some embodiments, it may be desirable to select apromoter based upon the desired plant cells to be transformed by thetransgene construct, the desired expression level of the transgene, thedesired tissue or subcellular compartment for transgene expression, thedevelopmental stage targeted, and the like. For example, aroot-preferred promoter may include, without limitation, a RolDpromoter, a RolD-2 promoter, a glycine rich protein promoter, a GRPpromoter, an ADH promoter, a maize ADH1 promoter, a PHT promoter, a Pht1gene family promoter, a metal uptake protein promoter, a maizemetallothionein protein promoter, a 35S CaMV domain A promoter, a pDJ3Spromoter, an SIREO promoter, a pMe1 promoter, an Sad1 promoter, an Sad2promoter, a TobRB7 promoter, an RCc3 promoter, an FaRB7 promoter, anSPmads promoter, an IDS2 promoter, a pyk10 promoter, an Lbc3leghemoglobin promoter, a PEPC promoter, a Gns1 glucanase root promoter,a 35S.sup.2promoter, a GI4 promoter, a GI5 promoter, and a GRP promoter

In addition to constitutive promoters, various inducible promotersequences may be employed in cases where it is desirable to regulatetransgene expression as the transgenic plant regenerates, matures,flowers, etc. Examples of such inducible promoters include promoters ofheat shock genes, protection responding genes (i.e., phenylalanineammonia lyase; see, for example Bevan et al., 1989, EMBO J. 8(7):899-906), wound responding genes (i.e., cell wall protein genes),chemically inducible genes (i.e., nitrate reductase, chitinase) and darkinducible genes (i.e., asparagine synthetase; see, for example U.S. Pat.No. 5,256,558). Also, a number of plant nuclear genes are activated bylight, including gene families encoding the major chlorophyll a/bbinding proteins (cab) as well as the small subunit ofribulose-1,5-bisphosphate carboxylase (rbcS) (see, for example, Tobinand Silverthorne, 1985, Annu Rev. Plant Physiol. 36: 569-593; Dean etal., 1989, Annu Rev. Plant Physiol. 40: 415-439.).

Other inducible promoters include ABA- and turgor-inducible promoters,the auxin-binding protein gene promoter (Schwob et al., 1993, Plant J.4(3): 423-432), the UDP glucose flavonoid glycosyl-transferase genepromoter (Ralston et al., 1988, Genetics 119(1): 185-197); the MPIproteinase inhibitor promoter (Cordero et al., 1994, Plant J. 6(2):141-150), the glyceraldehyde-3-phosphate dehydrogenase gene promoter(Kohler et al., 1995, Plant Mol. Biol. 29(6): 1293-1298; Quigley et al.,1989, J. Mol. Evol. 29(5): 412-421; Martinez et al., 1989, J. Mol. Biol.208(4): 551-565) and light inducible plastid glutamine synthetase genefrom pea (U.S. Pat. No. 5,391,725; Edwards et al., 1990, supra).

For a review of plant promoters used in plant transgenic planttechnology, see Potenza et al., 2004, In Vitro Cell. Devel. Biol—Plant,40(1): 1-22. For a review of synthetic plant promoter engineering, see,for example, Venter, M., 2007, Trends Plant Sci., 12(3): 118-124.

In certain embodiments, a 3′ transcription termination sequence is alsoincorporated downstream of the transgene in order to direct thetermination of transcription and permit correct polyadenylation of themRNA transcript. Suitable transcription terminators are those which areknown to function in plants, including without limitation, the nopalinesynthase (NOS) and octopine synthase (OCS) genes of Agrobacteriumtumefaciens, the T7 transcript from the octopine synthase gene, the 3′end of the protease inhibitor I or II genes from potato or tomato, theCaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator.In addition, a gene's native transcription terminator may be used. Inspecific embodiments, described by way of the Examples, infra, thenopaline synthase transcription terminator is employed.

Selectable Markers:

Selectable markers are typically included in transgene expressionvectors in order to provide a means for selecting plant transformants.While various types of markers are available, various negative selectionmarkers are typically utilized, including those which confer resistanceto a selection agent that inhibits or kills untransformed cells, such asgenes which impart resistance to an antibiotic (such as kanamycin,gentamycin, anamycin, hygromycin and hygromycinB) or resistance to aherbicide (such as sulfonylurea, gulfosinate, phosphinothricin andglyphosate). Screenable markers include, for example, genes encoding.beta.-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405),genes encoding luciferase (Ow et al., 1986, Science 234: 856-859) andvarious genes encoding proteins involved in the production or control ofanthocyanin pigments (See, for example, U.S. Pat. No. 6,573,432). The E.coli glucuronidase gene (gus, gusA or uidA) has become a widely usedselection marker in plant transgenics, largely because of theglucuronidase enzyme's stability, high sensitivity and ease of detection(e.g., fluorometric, spectrophotometric, various histochemical methods).Moreover, there is essentially no detectable glucuronidase in mosthigher plant species.

Transformation Methodologies and Systems:

Various methods for introducing the transgene expression vectorconstructs of the present disclosure into a plant or plant cell are wellknown to those skilled in the art, and any capable of transforming thetarget plant or plant cell may be utilized.

Agrobacterium-mediated transformation is perhaps the most common methodutilized in plant transgenics, and protocols for Agrobacterium-mediatedtransformation of a large number of plants are extensively described inthe literature (see, for example, Agrobacterium Protocols, Wan, ed.,Humana Press, 2.sup.nd edition, 2006). Agrobacterium tumefaciens is aGram negative soil bacteria that causes tumors (Crown Gall disease) in agreat many dicot species, via the insertion of a small segment oftumor-inducing DNA (“T-DNA”, ‘transfer DNA’) into the plant cell, whichis incorporated at a semi-random location into the plant genome, andwhich eventually may become stably incorporated there. Directly repeatedDNA sequences, called T-DNA borders, define the left and the right endsof the T-DNA. The T-DNA can be physically separated from the remainderof the Ti-plasmid, creating a ‘binary vector’ system.

Agrobacterium transformation may be used for stably transforming dicots,monocots, and cells thereof (Rogers et al., 1986, Methods Enzymol., 118:627-641; Hernalsteen et al., 1984, EMBO J., 3: 3039-3041; Hoykass-VanSlogteren et al., 1984, Nature, 311: 763-764; Grimsley et al., 1987,Nature 325: 167-1679; Boulton et al., 1989, Plant Mol. Biol. 12: 31-40;Gould et al., 1991, Plant Physiol. 95: 426-434). Various methods forintroducing DNA into Agrobacteria are known, including electroporation,freeze/thaw methods, and triparental mating. The most efficient methodof placing foreign DNA into Agrobacterium is via electroporation (Wiseet al., 2006, Three Methods for the Introduction of Foreign DNA intoAgrobacterium, Methods in Molecular Biology, vol. 343: AgrobacteriumProtocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, N.J.,pp. 43-53). In addition, given that a large percentage of T-DNAs do notintegrate, Agrobacterium-mediated transformation may be used to obtaintransient expression of a transgene via the transcriptional competencyof unincorporated transgene construct molecules (Helens et al., 2005,Plant Methods 1:13).

A large number of Agrobacterium transformation vectors and methods havebeen described (Karimi et al., 2002, Trends Plant Sci. 7(5): 193-5), andmany such vectors may be obtained commercially (for example, Invitrogen,Carlsbad, Calif.). In addition, a growing number of “open-source”Agrobacterium transformation vectors are available (for example, pCambiavectors; Cambia, Can berra, Australia). See, also, subsection herein onTRANSGENE CONSTRUCTS, supra. In a specific embodiment described furtherin the Examples, a pMON316-based vector was used in the leaf disctransformation system of Horsch et. al. (Horsch et al., 1995, Science227:1229-1231) to generate growth enhanced transgenic tobacco and tomatoplants.

Other commonly used transformation methods that may be employed ingenerating the transgenic plants of the present disclosure include,without limitation microprojectile bombardment, or biolistictransformation methods, protoplast transformation of naked DNA bycalcium, polyethylene glycol (PEG) or electroporation (Paszkowski etal., 1984, EMBO J. 3: 2727-2722; Potrykus et al., 1985, Mol. Gen. Genet.199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828; Shimamoto et al., 1989, Nature, 338: 274-276.

Biolistic transformation involves injecting millions of DNA-coated metalparticles into target cells or tissues using a biolistic device (or“gene gun”), several kinds of which are available commercially. Onceinside the cell, the DNA elutes off the particles and a portion may bestably incorporated into one or more of the cell's chromosomes (forreview, see Kikkert et al., 2005, Stable Transformation of Plant Cellsby Particle Bombardment/Biolistics, in: Methods in Molecular Biology,vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Pena, HumanaPress Inc., Totowa, N.J.).

Electroporation is a technique that utilizes short, high-intensityelectric fields to permeabilize reversibly the lipid bilayers of cellmembranes (see, for example, Fisk and Dandekar, 2005, Introduction andExpression of Transgenes in Plant Protoplasts, in: Methods in MolecularBiology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L.Pena, Humana Press Inc., Totowa, N.J., pp. 79-90; Fromm et al., 1987,Electroporation of DNA and RNA into plant protoplasts, in Methods inEnzymology, Vol. 153, Wu and Grossman, eds., Academic Press, London, UK,pp. 351-366; Joersbo and Brunstedt, 1991, Electroporation: mechanism andtransient expression, stable transformation and biological effects inplant protoplasts. Physiol. Plant. 81, 256-264; Bates, 1994, Genetictransformation of plants by protoplast electroporation. Mol. Biotech. 2:135-145; Dillen et al., 1998, Electroporation-mediated DNA transfer toplant protoplasts and intact plant tissues for transient gene expressionassays, in Cell Biology, Vol. 4, ed., Celis, Academic Press, London, UK,pp. 92-99). The technique operates by creating aqueous pores in the cellmembrane, which are of sufficiently large size to allow DNA molecules(and other macromolecules) to enter the cell, where the transgeneexpression construct (as T-DNA) may be stably incorporated into plantgenomic DNA, leading to the generation of transformed cells that cansubsequently be regenerated into transgenic plants.

Newer transformation methods include so-called “floral dip” methods,which offer the promise of simplicity, without requiring plant tissueculture, as is the case with all other commonly used transformationmethodologies (Bent et al., 2006, Arabidopsis thaliana Floral DipTransformation Method, Methods Mol Biol, vol. 343: AgrobacteriumProtocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, N.J.,pp. 87-103; Clough and Bent, 1998, Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana, Plant J.16: 735-743). However, with the exception of Arabidopsis, these methodshave not been widely used across a broad spectrum of different plantspecies. Briefly, floral dip transformation is accomplished by dippingor spraying flowering plants in with an appropriate strain ofAgrobacterium tumefaciens. Seeds collected from these T₀ plants are thengerminated under selection to identify transgenic T₁ individuals.Example 16 demonstrated floral dip inoculation of Arabidopsis togenerate transgenic Arabidopsis plants.

Other transformation methods include those in which the developing seedsor seedlings of plants are transformed using vectors such asAgrobacterium vectors. For example, such vectors may be used totransform developing seeds by injecting a suspension or mixture of thevector (i.e., Agrobacteria) directly into the seed cavity of developingpods (i.e., pepper pods, bean pods, pea pods and the like). Still othertransformation methods include those in which the flower structure istargeted for vector inoculation.

In addition, although transgenes are most commonly inserted into thenuclear DNA of plant cells, transgenes may also be inserted intoplastidic DNA (i.e., into the plastome of the chloroplast). In mostflowering plants, plastids do not occur in the pollen cells, andtherefore transgenic DNA incorporated within a plastome will not bepassed on through propagation, thereby restricting the trait frommigrating to wild type plants. Plastid transformation, however, is morecomplex than cell nucleus transformation, due to the presence of manythousands of plastomes per cell (as high as 10,000).

Transplastomic lines are genetically stable only if all plastid copiesare modified in the same way, i.e. uniformly. This is typically achievedthrough repeated regeneration under certain selection conditions toeliminate untransformed plastids, by segregating transplastomic anduntransformed plastids, resulting in the selection of homoplasmic cellscarrying the transgene construct and the selectable marker stablyintegrated therein. Plastid transformation has been successfullyperformed in various plant species, including tobacco, potatoes, oilseedrape, rice, and Arabidopsis.

To generate transplastomic lines carrying an Ω-amidase transgene, thetransgene expression cassette is inserted into flanking sequences fromthe plastome. Using homologous recombination, the transgene expressioncassette becomes integrated into the plastome via a naturalrecombination process. The basic DNA delivery techniques for plastidtransformation include particle bombardment of leaves or polyethyleneglycol-mediated DNA transformation of protoplasts. Transplastomic plantscarrying transgenes in the plastome may be expressed at very highlevels, due to the fact that many plastids (i.e., chloroplasts) percell, each carrying many copies of the plastome. This is particularlythe case in foliar tissue, where a single mature leaf cell may containover 10,000 copies of the plastome. Following a successfultransformation of the plastome, the transplastomic events carry thetransgene on every copy of the plastid genetic material. This can resultin the transgene expression levels representing as much as half of thetotal protein produced in the cell.

Plastid transformation methods and vector systems are described, forexample, in recent U.S. Pat. Nos. 7,528,292; 7,371,923; 7,235,711; and,7,193,131. See also U.S. Pat. Nos. 6,680,426 and 6,642,053.

The foregoing plant transformation methodologies may be used tointroduce at least one transgene into a number of different plant cellsand tissues, including without limitation, whole plants, tissue andorgan explants including chloroplasts, flowering tissues and cells,protoplasts, meristem cells, callus, immature embryos and gametic cellssuch as microspores, pollen, sperm and egg cells, tissue cultured cellsof any of the foregoing, any other cells from which a fertileregenerated transgenic plant may be generated. Callus is initiated fromtissue sources including, but not limited to, immature embryos, seedlingapical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation.

Methods of regenerating individual plants from transformed plant cells,tissues or organs are known and are described for numerous plantspecies.

As an illustration, transformed plantlets (derived from transformedcells or tissues) are cultured in a root-permissive growth mediumsupplemented with the selective agent used in the transformationstrategy. Once rooted, transformed plantlets are then transferred tosoil and allowed to grow to maturity. Upon flowering, the mature plantsare preferably selfed (self-fertilized), and the resultant seedsharvested and used to grow subsequent generations.

T₀ transgenic plants may be used to generate subsequent generations(e.g., T₁, T₂, etc.) by selfing of primary or secondary transformants,or by sexual crossing of primary or secondary transformants with otherplants (transformed or untransformed). During the mature plant growthstage, the plants are typically examined for growth phenotype, nitrogenuse efficiency, CO₂ fixation rate, etc. (see following subsection).

Selection of Transgenic Plants with Increased Nitrogen Use Efficiency:

Transgenic plants may be selected, screened and characterized usingstandard methodologies. The preferred transgenic plants of the presentdisclosure will exhibit one or more phenotypic characteristicsindicative of increased nitrogen use efficiency, including withoutlimitation, faster growth rates, greater seed and fruit/pod yields,earlier and more productive flowering, increased tolerance to high saltconditions, and increased biomass yields. Transgenic plants aretypically regenerated under selective pressure in order to selecttransformants prior to creating subsequent transgenic plant generations.In addition, the selective pressure used may be employed beyond T₀generations in order to ensure the presence of the desired transgeneexpression construct or cassette.

T₀ transformed plant cells, calli, tissues or plants may be identifiedand isolated by selecting or screening for the genetic composition ofand/or the phenotypic characteristics encoded by marker genes containedin the transgene expression construct used for the transformation. Forexample, selection may be conducted by growing potentially-transformedplants, tissues or cells in a growth medium containing a repressiveamount of antibiotic or herbicide to which the transforming geneticconstruct can impart resistance. Further, the transformed plant cells,tissues and plants can be identified by screening for the activity ofmarker genes (i.e., .beta.-glucuronidase) which may be present in thetransgene expression construct.

Various physical and biochemical methods may be employed for identifyingplants containing the desired transgene expression construct, as is wellknown. Examples of such methods include Southern blot analysis orvarious nucleic acid amplification methods (i.e., PCR) for identifyingthe transgene, transgene expression construct or elements thereof,Northern blotting, 51 RNase protection, reverse transcriptase PCR(RT-PCR) amplification for detecting and determining the RNAtranscription products, and protein gel electrophoresis, Westernblotting, immunoprecipitation, enzyme immunoassay, and the like may beused for identifying the protein encoded and expressed by the transgene.

In another approach, expression levels of genes, proteins and/ormetabolic compounds that are know to be modulated by transgeneexpression in the target plant may be used to identify transformants. Inone embodiment of the present disclosure, increased levels of the signalmetabolite 2-oxoglutaramate in leaf tissue, or decreased levels in theroot tissue, or a higher leaf-to-root ratio of 2-oxoglutaramate may beused to screen for desirable transformants.

Ultimately, the transformed plants of the present disclosure may bescreened for increased nitrogen use efficiency. Nitrogen use efficiencymay be expressed as plant yield per given amount of nitrogen. Indeed,some degree of phenotypic screening is generally desirable in order toidentify transformed lines with the fastest growth rates, the highestseed yields, etc., particularly when identifying plants for subsequentselfing, cross-breeding and back-crossing.

Various parameters may be used for this purpose, including withoutlimitation, growth rates, total fresh weights, dry weights, seed andfruit yields (number, weight), seed and/or seed pod sizes, seed podyields (e.g., number, weight), leaf sizes, plant sizes, increasedflowering, time to flowering, overall protein content (in seeds, fruits,plant tissues), specific protein content (i.e., Ω-amidase), nitrogencontent, free amino acid, and specific metabolic compound levels (i.e.,2-oxoglutaramate). Generally, these phenotypic measurements are comparedwith those obtained from a parental identical or analogous plant line,an untransformed identical or analogous plant, or an identical oranalogous wild-type plant (i.e., a normal or parental plant).Preferably, and at least initially, the measurement of the chosenphenotypic characteristic(s) in the target transgenic plant is done inparallel with measurement of the same characteristic(s) in a normal orparental plant. Typically, multiple plants are used to establish thephenotypic desirability and/or superiority of the transgenic plant inrespect of any particular phenotypic characteristic.

Preferably, initial transformants are selected and then used to generateT₁ and subsequent generations by selfing (self-fertilization), until thetransgene genotype breeds true (i.e., the plant is homozygous for thetransgene). In practice, this is accomplished by screening at eachgeneration for the desired traits and selfing those individuals, oftenrepeatedly (i.e., 3 or 4 generations). As exemplified herein, transgenicplant lines propagated through at least one sexual generation (Tobacco,Arabidopsis, Tomato) demonstrated higher transgene product activitiescompared to lines that did not have the benefit of sexual reproductionand the concomitant increase in transgene copy number.

Stable transgenic lines may be crossed and back-crossed to createvarieties with any number of desired traits, including those withstacked transgenes, multiple copies of a transgene, etc. Various commonbreeding methods are well known to those skilled in the art (see, e.g.,Breeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987)). Additionally, stabletransgenic plants may be further modified genetically, by transformingsuch plants with further transgenes or additional copies of the parentaltransgene. Also contemplated are transgenic plants created by singletransformation events which introduce multiple copies of a giventransgene or multiple transgenes.

Nitrogen use efficiency may be expressed as plant yield per given amountof nitrogen.

Methods for Increasing Nitrogen Use Efficiency:

Certain aspects of the present disclosure relate to methods forincreasing nitrogen use efficiency of a plant.

One particular aspect relates to a method for increasing nitrogen useefficiency of a plant relative to a wild type or untransformed plant ofthe same species, by: (a) introducing an Ω-amidase transgene into theplant, where the Ω-amidase transgene is operably linked to aroot-preferred promoter; (b) expressing the Ω-amidase transgene in roottissue of the plant or the progeny of the plant; and (c) selecting aplant having an increased leaf-to-root ratio of 2-oxoglutaramaterelative to a plant of the same species that does not contain anΩ-amidase transgene, where the increased leaf-to-root ratio of2-oxoglutaramate results in increased nitrogen use efficiency.

In certain embodiments, the Ω-amidase transgene encodes a polypeptidehaving an amino acid sequence that is at least 90% identical to an aminoacid sequence encoded by a polypeptide selected from AAL91613.1,ACN30911.1, ABK22312.1, ACJ85250.1, AAQ97821.1, CBJ25483.1, EFN54567.1,NP_(—)196765.2, XP_(—)002871509.1, NP_(—)974769.1, XP_(—)002309478.1,XP_(—)002279687.1, NP_(—)001146676.1, NP_(—)001146295.1,NP_(—)001049134.1, XP_(—)002516116.1, XP_(—)001766085.1,XP_(—)001756522.1, XP_(—)002969787.1, XP_(—)002985119.1,XP_(—)002948137.1, XP_(—)001690839.1, NP_(—)001057093.1,XP_(—)002468410.1, NP_(—)064587.1, XP_(—)001089575.2, XP_(—)001502234.1,XP_(—)002502298.1, XP_(—)526254.2, XP_(—)535718.2, XP_(—)002716659.1,NP_(—)001033222.1, NP_(—)001029298.1, NP_(—)001016633.1,NP_(—)001085409.1, XP_(—)002758928.1, XP_(—)003064056.1,NP_(—)001135127.1, XP_(—)001622809.1, NP_(—)991174.2, XP_(—)002594716.1,NP_(—)075664.1, XP_(—)001370849.1, NP_(—)001090454.1, XP_(—)002999170.1,XP_(—)002917137.1, XP_(—)002741281.1, XP_(—)002131764.1, NP_(—)594154.1,XP_(—)001742101.1, XP_(—)416604.2, XP_(—)002194275.1, XP_(—)001599587.1,XP_(—)002410555.1, XP_(—)003035898.1, XP_(—)002183613.1,XP_(—)001875493.1, XP_(—)002112209.1, XP_(—)636983.1, XP_(—)002158547.1,XP_(—)002839272.1, XP_(—)307722.3, XP_(—)001819629.1, XP_(—)001268376.1,ZP_(—)08115581.1, YP_(—)001320997.1, XP_(—)369268.1, XP_(—)002626458.1,XP_(—)751200.1, XP_(—)001657673.1, XP_(—)002173486.1, XP_(—)001212538.1,XP_(—)001258462.1, XP_(—)002434512.1, XP_(—)960906.1, XP_(—)002847679.1,XP_(—)967861.1, XP_(—)002426154.1, XP_(—)003176259.1, XP_(—)500602.1,XP_(—)001428419.1, XP_(—)003014235.1, XP_(—)001393123.1,ZP_(—)03608460.1, XP_(—)002147261.1, ZP_(—)03293831.1,XP_(—)002290043.1, XP_(—)003065597.1, XP_(—)001588734.1,YP_(—)001273073.1, XP_(—)001552800.1, XP_(—)446414.1, XP_(—)002792830.1,XP_(—)001998501.1, YP_(—)003780301.1, NP_(—)013455.1, XP_(—)002404736.1,YP_(—)001086961.1, ZP_(—)05328587.1, ZP_(—)05399936.1,YP_(—)001113615.1, XP_(—)001247884.1, XP_(—)390426.1, XP_(—)003025334.1,XP_(—)002052999.1, YP_(—)769862.1, ZP_(—)07325748.1, ZP_(—)05349666.1,YP_(—)471237.1, YP_(—)002977603.1, YP_(—)001202760.1, ZP_(—)07592670.1,and NP_(—)386723.1. In other embodiments, the Ω-amidase transgene isincorporated into the genome of the plant. In still other embodiments,the root-preferred promoter is selected from RolD promoter, RolD-2promoter, glycine rich protein promoter, GRP promoter, ADH promoter,maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metaluptake protein promoter, maize metallothionein protein promoter, 35SCaMV domain A promoter. pDJ3S promoter, SIREO promoter, pMe1 promoter,Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter,35S.sup.2promoter, GI4 promoter, GI5 promoter, and GRP promoter.

In other embodiments, endogenous Ω-amidase expression in leaf tissue isinhibited. In still other embodiments, the endogenous Ω-amidaseexpression in leaf tissue is inhibited by recessive gene disruption,dominant gene silencing, or a chemical inhibitor. In yet otherembodiments, the endogenous Ω-amidase expression in leaf tissue isinhibited by a recessive gene disruption selected from a mutantΩ-amidase gene that eliminates endogenous Ω-amidase expression, anendogenous Ω-amidase knockout mutant, and an endogenous Ω-amidaseknockdown mutant. In further embodiments, the endogenous Ω-amidaseexpression in leaf tissue is inhibited by an RNAi antisenseoligonucleotide that is specific for an endogenous Ω-amidase gene. Instill further embodiments, the endogenous Ω-amidase expression in leaftissue is inhibited by a chemical inhibitor selected from6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropylfluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate,silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.

In other embodiments, the leaf-to-root ratio of 2-oxoglutaramate is atleast two times higher than that of a progenitor or wild type plant ofthe same species. In still other embodiments, the plant further containsa GPT transgene. In yet other embodiments, the GPT transgene is aGPT/F:V mutant given by SEQ ID NO:1. In further embodiments, the plantfurther comprises a GPT transgene and a GS transgene. In otherembodiments, the GPT transgene and GS transgene are each operably linkedto a leaf-preferred promoter. In still further embodiments, endogenousGPT expression in the plant is increased by gene activation. In yetother embodiments, endogenous GS expression in the plant is increased bygene activation. In other embodiments, each transgene is codon optimizedfor expression in the plant.

Another aspect relates to a method for increasing nitrogen useefficiency of a plant relative to a wild type or untransformed plant ofthe same species, by: (a) inhibiting endogenous Ω-amidase expression inleaf tissue of the plant; and (b) selecting a plant having an increasedleaf-to-root ratio of 2-oxoglutaramate relative to a plant of the samespecies that does not have inhibited endogenous Ω-amidase expression inleaf tissue, where the increased leaf-to-root ratio of 2-oxoglutaramateresults in increased nitrogen use efficiency.

In certain embodiments, the endogenous Ω-amidase expression in leaftissue is inhibited by recessive gene disruption, dominant genesilencing, or a chemical inhibitor. In other embodiments, the endogenousΩ-amidase expression in leaf tissue is inhibited by a recessive genedisruption selected from a mutant Ω-amidase gene that eliminatesendogenous Ω-amidase expression, an endogenous Ω-amidase knockoutmutant, and an endogenous Ω-amidase knockdown mutant. In still otherembodiments, the endogenous Ω-amidase expression in leaf tissue isinhibited by an RNAi antisense oligonucleotide that is specific for anendogenous Ω-amidase gene. In yet other embodiments, the chemicalinhibitor selected from 6-diazo-5-oxo-nor-leucine,p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide,phenylmercuriacetate, Iodoacetate, silver nitrate,chloromercuricphenylsulfonic acid, and copper sulfate.

In other embodiments, the plant further contains an Ω-amidase transgene,wherein the Ω-amidase transgene is operably linked to a root-preferredpromoter. In still other embodiments, the leaf-to-root ratio of2-oxoglutaramate is at least two times higher than that of a progenitoror wild type plant of the same species. In yet other embodiments, theplant further contains a GPT transgene. In further embodiments, the GPTtransgene is a GPT/F:V mutant given by SEQ ID NO:1. In still furtherembodiments, the plant further contains a GPT transgene and a GStransgene. In other embodiments, the GPT transgene and GS transgene areeach operably linked to a leaf-preferred promoter. In yet furtherembodiments, endogenous GPT expression in the plant is increased by geneactivation. In other embodiments, endogenous GS expression in the plantis increased by gene activation. In still other embodiments, eachtransgene is codon optimized for expression in the plant.

In further embodiments of the methods for increasing nitrogen useefficiency of a plant, the plant may contain other transgenes known toincrease nitrogen utilization efficiency, including without limitationthose described in U.S. Pat. No. 7,560,626.

In the Examples provided herein, the transgene and control plants allreceived the same nutrient solutions in the same amounts. The transgenicplants were consistently characterized by higher yields, and thus havehigher nitrogen use efficiencies.

EXAMPLES Example 1 Effects of Increasing Expression Levels of GS and GPTon OMEGA-Amidase Pathway

Materials and Methods:

Generation of Transgenic Plants: Plants were genetically engineered toover-produce 2-oxoglutaramate by over expressing GS and GPT transgenes,as described in U.S. patent application Ser. No. 12/551,271. Theresulting phenotypic effects were evaluated. Three sets of transgenictobacco lines were generated: one set over-expressing GPT to increaseGRMT catalytic capacity; a second set over-expressing GS to increase, inthe leaves only, the catalytic capacity to make the glutamine substrateof GPT; and a third set over expressing GS and GPT, produced by sexuallycrossing fast-growing progeny of the single transgene lines.

Growth of Engineered Tobacco and Arabidopsis: Wild type and engineeredtobacco seeds were surface sterilized and germinated in phytotrayscontaining M&S medium. The medium for the engineered plants containedkanamycin (10 ug/ml). The vigorously growing seedlings were transferredat 17 d to a sand culture, covered to control humidity for 4 d to aidadaptation to ambient conditions; plants were continuously provided anutrient solution (Knight and Langston-Unkefer, 1988, Science 241: 951)containing 10 mM KNO₃. The growth conditions were as describedpreviously (Knight and Langston-Unkefer, supra). Tissue was harvestedbetween 32-35 d after the transplant unless the plants were being grownfor seed production. Wild type and engineered Arabidopsis seeds weresurface sterilized and germinated in phytotrays containing M&S medium.For the engineered plants the medium contained kanamycin (10 ug/ml).

The seedlings were transferred to the ArabiSystem using the Promix(Lehle Seeds) growth medium and grown at 24.degree. C. with 16 h lightand 8 h dark periods. Plants were grown to maturity.

Results:

The over-expression of GS in tobacco generated two classes of progeny;those that grew faster and over-expressed GS only in their leaves, thusincreasing their leaf-to ratio of GS, and a second class that grew atnormal rates and over-expressed GS in both leaves and roots, therebymaintaining a normal leaf-to-root ratio. Regulation of GS and GPTexpression in these plants appears complex and expression of each geneappears to influence the other (see Tables 2 and 3); over-expression ofonly GPT in leaves and roots was accompanied by increased GS activity inthe leaf and only normal GS activity in the root. Increased GSexpression only in the leaf was accompanied by increased GPT activity inthe leaf and lower GPT activity in the root. These responses wereevident in the GS+GPT transgenic plants as well. Over-expression ofeither GS or GPT was accompanied by lower Ω-amidase activity in theleaves and greater Ω-amidase activity in the roots. GPT and GS+GPTtransgenic plants showed the largest increases in root Ω-amidaseactivity. These plants responded to expression of the transgenes byaltering their Ω-amidase activities such that they tend to increase theleaf root 2-oxoglutaramate pool, and maintain the root 2-oxoglutaramatepool. These responses combined in the GS+GPT over-expressing plants togenerate the highest leaf and lowest root 2-oxoglutaramate pools and thehighest leaf and lowest GS and GPT activities.

Tables 2, 3, and 4 below depict the effects of engineering for greater2-oxoglutaramate (2-0GM) biosynthesis. Tables 2 and 3 depict resultsfrom tobacco plants, and Table 4 depicts results from Arabidopsisthaliana plants.

TABLE 2 Effects of engineering greater 2-oxoglutaramate biosynthesiscapability Leaf Root 2-OGM Leaf Leaf Leaf Amidase Root GS Root RootAmidase 2-OGM nmol/ GS GPT nmol/ umol/ GPT nmol/ Tobacco nmol/ gfwtumol/ nmol/ gfwt/h gfwt/m nmol/ gfwt/h Genotype gfwt (Leaf/Root) gfwt/mgfwt/h (GPT/Amidase) (Leaf/Root) gfwt/h (GPT/Amidase) Wild type 191 116(1.6) 7.8 100 191 (0.5) 2.1 (3.7) 236 252 (0.9) +GPT 384 143 (2.7) 10.5196 118 (1.7) 1.9 (5.5) 566 440 (1.3) +GS 502 131 (3.8) 11.6 288 112(2.6) 1.7 (6.8) 136 372 (0.4) +GS + GPT 701  80 (8.7) 16.3 731 149 (4.9)1.8 (9.1) 117 292 (0.4)

TABLE-US-00005 TABLE 2 Effects of engineering greater 2-oxoglutaramatebiosynthesis capability Root Leaf Amidase Root GS 2-OGM Leafnmol/umol/Root Root Amidase Leaf nmol/gfwt Leaf GS GPT gfwt/h gfwt/m GPTnmol/gfwt/h Tobacco 2-OGM (Leaf/umol/nmol/(GPT/(Leaf/nmol/(GPT/Genotypenmol/gfwt Root) gfwt/m gfwt/h Amidase) Root) gfwt/h Amidase) Wild type191 116 (1.6) 7.8 100 191 (0.5) 2.1 (3.7) 236 252 (0.9)+GPT 384 143(2.7) 10.5 196 118 (1.7) 1.9 (5.5) 566 440 (1.3)+GS 502 131 (3.8) 11.6288 112 (2.6) 1.7 (6.8) 136 372 (0.4)+GS+GPT 701 80 (8.7) 16.3 731 149(4.9) 1.8 (9.1) 117 292 (0.4)

In Table 2 above, “gfwt” refers to grams fresh weight and “nmol/gfwt/h”refers to nano moles per grams fresh weight per hour.

TABLE 3 Effects of engineering greater 2-oxoglutaramate biosynthesiscapability NO₃ CO₂ Seed Whole Tobacco uptake rate Leaf NO₃ Root NO₃ LeafProtein Chlorophyll Fixed Rate RGR yield Plant Genotype mm/gfwthμmol/gfwt μmol/gfwt Mg/gfwt μg/gfwt mm/m²/s Mg/g/d g/plt gfwt Wild type 4.3 ± 2.8 69.3 ± 4.9 29.9 ± 2.2 4.3  818 ± 10 7.7 226 1.0  21.0 (100%)(100%) +GPT 10.9 ± 2.4 77.6 ± 8.2 57.5 ± 6.0 5.2 1044 ± 3 12.9 ND NM 31 (120%) (147%) +GS 11.3 ± 1.9 22.8 ± 2.7 12.1 ± 3.2 6.9 1109 ± 6 13.5 269NM 35.6 (160%) (169%) +GS + GPT 19.5 ± 3.1 51.1 ± 1.8 30.1 ± 3.5 7.3 1199 ± 11 20.6 346 2.87 71.9 (170%) (342%)

TABLE 4 Arabidopsis engineered to over-produce 2-oxoglutaramate WholeLeaf Leaf Leaf Leaf Plant GS GPT 2-OGM Protein Fresh ArabidopsisActivity Activity nmol · mg/gfwt Wt. g Genotype μmol/gfwt. m nmol/gfwt/hgfwt/h (% wt) (% wt) Wild type 6.89 184 184 6.06* 0.246 +GS + GPT 18.71077 395.5 7.46* 1.106 (123%) (449%)

Example 2 Plant Expression Vector Modulating Root Ω-Amidase ExpressionLevels

OMEGA-amidase expression levels are increased in root tissues bygenerating transgenic plants transformed with expression constructscontaining an Ω-amidase coding sequence, including but not limited toany of the Ω-amidase coding sequences disclosed herein, under thecontrol of a root-preferred promoter. It is believed that increasedlevels of Ω-amidase in root tissues result in increased breakdown of thesignal metabolite 2-oxoglutaramate.

A construct for transforming plants includes an expression cassetteencoding a suitable root-preferred promoter, a sequence encoding a plantΩ-amidase, and a terminator sequence. In this example, the expressioncassette contains the glycine-rich protein (GRP) promoter (Goddemeier etal., 1998, Plant Mol. Biol. 36(5): 799-802), the Arabidopsis thalianaΩ-amidase coding sequence, and the NOS terminator. The GRP promotersequence is shown below [SEQ ID NO: 16]:

GAAATTAAACCCAGGGTCGACAGCGCCCACTATAGAGAAAAAATTGAAATGTTTTGAGAATCGGATGATTTTTTTTAACTATTAGGTCTAGTTTGAAAACCCTATTTTCTAACAAAGGGATTTTCATTTTTATAAGAGAAAATAAACTAACTTTTCTTGAGAAAATAAAATTCTTTGGAAAAATGGATTTCTCAAACTAGCTCTTACGGCTAGTTTGGAAACCCCAATTTCACACGGGATTCTCATTTTCCCAAGGGAAAAATGAACTAATTTCCCTTAGAAAAATGAGAATCCCGTGGGAAATTGGGATTTTCAAAGTAGCCCTTATAGTGGAAATAAGTTATGGTGTCTCGCTCGTATGGTTATGTAGGGCCGCGCGTGTATTCCAGCGCCGGCCGCATGGATACCCTATCGATTCTGACTTCTCTGTCTCAGGAAAATAATACAGCCACGATTAACGGAACCTGCTGGCTGGATCCATGATTACTCACTTGACTTCACATCGATCCAAATTATCTAGCTTGCACGTTCATGGGTCGCCTCGCTCGCCCGATCGATATTACGTACACCATAGATTAGTACTATATGGAGTGGAGTGTTGAATGGATGCTCTTTATTATTCTAGCCAAGTTATCAAGCCGGGCACTTGCATCGGAAGGAGTACCAGTGTACGCATCAGATCAGACGATAATCGATCAAGATGGGTACGAGATTTGCCGCTTGCTTCCTGTTCTTGATGGGCAATCTTTTCGGGCCTTGAACGTCGGAGAATCGACTATACGAAATCCTAGGTCAACTATACATTGGTTGATGCTTCCGTGTAGTTTTACCAGTTCATCGGTCTCTAGCTTGTTGTTTGCGACGACTTCACGTGGCCACGCGTTTACTGCGCTCTGCTCAAAGAAATTGCCTACAGTGCCTGGCGTCAGCTGCAGGCGTTGAATCCGAGGTCGCGCGCCGCAGAATAAGTACGAGTCAAAGGCTGAGCTGCATGCCGTACCGGCCTTTATTAATAGCTGAGCTCTACTCGCTACGTCAGTATAGTATAGCACGGTCATATATATACTATAGCTATAGCTGTGGGGTACCGTGTCCGTATCGTGAATCTGAAGTCGAACAGTGATATGGCGTACTATCTAATAATGTCCCGTGCAGTAATATCACTGTTGCCGACGATGGGAATCTCTAGTTTTGACAGAAACCAAAGCAACTGCTAGCTAATTAATTCCAGAGAGATCGATTTCTACAGTGCTGCAACAATCAATGCAATTGGCATCAGACGATATATGCTAATGGTTTCTTTATCGATACGTGGTCAACAGAGCTCTCTCGCCCGCCCTGATCAGATCTCATCGCACATGGACACCCATCTGCCAACCCAACACGGGCGGGGGAACCACCGTGAAACATCGCGTTCATGCACGACCCCCCCGCAGGCCGCAGCTATAAATACCCATGCAATGCAATGCAGCGGGTCATCATCGACTCCACCTGGACTCGCTCAC TGGCAATGGCTACCACCAGC

Alternatively, an expression cassette containing the rolD promoter andthe Arabidopsis thaliana Ω-amidase coding sequence may be used. Thesequence of this construct is shown below: (ATG start codon of theΩ-amidase gene shown in bold) [SEQ ID NO: 17]:

GACGTCGGTACCGAATTTGTTCGTGAACTATTAGTTGCGGGCCTTGGCATCCGACTACCTCTGCGGCAATATTATATTCCCTGGGCCCACCGTGAACCCAATTTCGCCTATTTATTCATTACCCCCATTAACATTGAAGTAGTCATGATGGGCCTGCAGCACGTTGGTGAGGCTGGCACAACTCATCCATATACTTTCTGACCGGATCGGCACATTATTGTAGAAAACGCGGACCCACAGCGCACTTTCCAAAGCGGTGCCGCGTCAGAATGCGCTGGCAGAAAAAAATTAATCCAAAAGTACCCTCCAAGCAGCCCATATAAACGCGTTTACAAATCCGCTAACCTCAACAATTTGAGCAGAGAAAATTCGCACCTACAAGGCAGATGGCATCATCATTCAATCCAGAGCAGGCAAGAGTTCCTTCAGCATTACCTTTACCAGCACCACCACTTACCAAATTCAACATCGGACTTTGTCAATTGAGTGTTACTTCTGATAAGAAAAGAAACATTTCACATGCTAAGAAAGCAATCGAAGAGGCTGCTAGTAAGGGAGCTAAACTCGTTCTTTTGCCTGAAATATGGAACTCACCATACAGTAACGATTCTTTTCCTGTGTACGCAGAAGAGATCGATGCTGGAGGTGATGCATCTCCATCAACTGCTATGCTCTCAGAAGTTAGTAAGAGACTCAAGATTACAATTATCGGAGGTTCAATTCCTGAGAGAGTTGGAGATAGGTTGTATAACACATGTTGCGTGTTCGGATCTGATGGAGAGCTCAAGGCTAAGCATAGGAAGATTCACCTCTTCGATATAGATATTCCTGGAAAGATCACCTTCATGGAATCAAAAACACTTACCGCTGGAGAGACTCCAACAATTGTTGATACAGATGTGGGTAGAATCGGAATAGGTATATGTTACGATATCAGGTTCCAAGAATTGGCTATGATATATGCTGCAAGAGGAGCACATCTCTTATGCTACCCTGGAGCTTTCAATATGACTACAGGTCCATTGCACTGGGAGCTTTTGCAAAGAGCTAGGGCAACAGATAACCAGCTCTATGTTGCTACCTGCTCTCCTGCAAGAGATTCAGGAGCTGGTTACACCGCATGGGGTCATTCTACTCTTGTTGGACCATTTGGTGAAGTGTTGGCTACCACTGAGCACGAAGAGGCTATTATAATCGCAGAAATCGATTACAGTATACTTGAGCAGAGAAGGACTTCTCTCCCATTAAATAGGCAGAGGAGGGGTGATTTATACCAGTTAGTTGATGTTCAGAGATTAGATAGTAAGTGACACGTGTGAATTACAGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGA TCGGGGGTACCGACGTC

For transformation of plants, the expression cassette, above, is clonedinto a suitable vector. For Agrobacterium mediated transformation, theabove construct is cloned into the TF101.1 vector, which carries thespectinomycin resistance selectable marker gene.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Example 3 Increased Growth of Transgenic Alfalfa Plants CarryingRoot-Preferred OMEGA-Amidase Transgene

In this example, alfalfa plant growth was increased by introducing anΩ-amidase transgene under the control of a highly root-preferredpromoter. The resulting transgenic alfalfa plants showed decreased2-oxoglutaramate concentration in roots, increased leaf-to-root ratio of2-oxoglutaramate, and enhanced growth relative to wild type alfalfaplants. Alfalfa plants (Medicago sativa, var Ladak) were transformedwith the Arabidopsis Ω-amidase coding sequence truncated to remove thechloroplast transit peptide, under the control of a truncatedAgrobacterium rhizogenes RolD promoter within the expression vectorpTF101.1.

Materials and Methods:

Agrobacterium Vectors: The expression vector pTF101.1 was engineered tocarry the Ω-amidase transgene expression cassette of SEQ ID NO: 39 (RolDpromoter+Ω-amidase+NOS terminator) and was transferred to Agrobacteriumtumefaciens strain LBA4404 cultures using a standard electroporationmethod (McCormac et al., 1998, Molecular Biotechnology 9:155-159). Thetruncated Agrobacterium rhizogenes RolD promoter utilized is the pD-02isoform (RolD2) described in Leach and Aoyagi, 1991, Plant Sci. 79,69-76. Leach and Aoyagi describe the RolD2 promoter as being a highlyroot-preferred promoter that drove high levels of expression in roottissue. Transformed Agrobacterium were selected on media containing 50.mu.g/ml of chloroamphenicol. Transformed Agrobacterium cells were grownin LB culture media containing 25 .mu.g/ml of antibiotic for 36 hours.At the end of the 36 hr growth period cells were collected bycentrifugation and cells from each transformation were resuspended in100 ml LB broth without antibiotic.

The nucleotide sequence of the pTF101.1vector+rolD-02promoter+Arabidopsis Ω-amidase (codon optimized forArabidopsis)+nos terminator (SEQ ID NO: 39) is set forth below.

Underlined nucleotides=rolD-02 promoter, bold nucleotides=Ω-amidasecoding region, italicized nucleotides include the nos terminator region(and some Cambia vector sequence), and other nucleotides=pTF101.1vector.

AGTACTTTAAAGTACTTTAAAGTACTTTAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAATAATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCTAATCGCTTGAGTTAACGCCGGCGAAGCGGCGTCGGCTTGAACGAATTTCTAGCTAGACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAAATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCCCATGATGTTTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACCCCAAAAAAACATGTCATAACAAGAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGACGGCAGTTACGCTACTTGCATTACAGCTTACGAACCGAACGAGGCTTATGTCCACTGGGTTCGTGCCCGAATTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTAATTCTTCAAGACGTGCTCAAATCACTATTTCCACACCCCTATATTTCTATTGCACTCCCTTTTAACTGTTTTTTATTACAAAAATGCCCTGGAAAATGCACTCCCTTTTTGTGTTTGTTTTTTTGTGAAACGATGTTGTCAGGTAATTTATTTGTCAGTCTACTATGGTGGCCCATTATATTAATAGCAACTGTCGGTCCAATAGACGACGTCGATTTTCTGCATTTGTTTAACCACGTGGATTTTATGACATTTTATATTAGTTAATTTGTAAAACCTACCCAATTAAAGACCTCATATGTTCTAAAGACTAATACTTAATGATAACAATTTTCTTTTAGTGAAGAAAGGGATAATTAGTAAATATGGAACAAGGGCAGAAGATTTATTAAAGCCGCGTAAGAGACAACAAGTAGGTACGTGGAGTGTCTTAGGTGACTTACCCACATAACATAAAGTGACATTAACAAACATAGCTAATGCTCCTATTTGAATAGTGCATATCAGCATACCTTATTACATATAGATAGGAGCAAACTCTAGCTAGATTGTTGAGAGCAGATCTCGGTGACGGGCAGGACCGGACGGGGCGGTACCGGCAGGCTGAAGTCCAGCTGCCAGAAACCCACGTCATGCCAGTTCCCGTGCTTGAAGCCGGCCGCCCGCAGCATGCCGCGGGGGGCATATCCGAGCGCCTCGTGCATGCGCACGCTCGGGTCGTTGGGCAGCCCGATGACAGCGACCACGCTCTTGAAGCCCTGTGCCTCCAGGGACTTCAGCAGGTGGGTGTAGAGCGTGGAGCCCAGTCCCGTCCGCTGGTGGCGGGGGGAGACGTACACGGTCGACTCGGCCGTCCAGTCGTAGGCGTTGCGTGCCTTCCAGGGGCCCGCGTAGGCGATGCCGGCGACCTCGCCGTCCACCTCGGCGACGAGCCAGGGATAGCGCTCCCGCAGACGGACGAGGTCGTCCGTCCACTCCTGCGGTTCCTGCGGCTCGGTACGGAAGTTGACCGTGCTTGTCTCGATGTAGTGGTTGACGATGGTGCAGACCGCCGGCATGTCCGCCTCGGTGGCACGGCGGATGTCGGCCGGGCGTCGTTCTGGGCTCATGGTAGATCCCCCGTTCGTAAATGGTGAAAATTTTCAGAAAATTGCTTTTGCTTTAAAAGAAATGATTTAAATTGCTGCAATAGAAGTAGAATGCTTGATTGCTTGAGATTCGTTTGTTTTGTATATGTTGTGTTGAGAATTAATTCTCGAGGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGTAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATAAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAATTGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTCAACGATGGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATAAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAATTGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTGACCTGCAGGCATGCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGTCGGTACCGAATTTGTTCGTGAACTATTAGTTGCGGGCCTTGGCATCCGACTACCTCTGCGGCAATATTATATTCCCTGGGCCCACCGTGAACCCAATTTCGCCTATTTATTCATTACCCCCATTAACATTGAAGTAGTCATGATGGGCCTGCAGCACGTTGGTGAGGCTGGCACAACTCATCCATATACTTTCTGACCGGATCGGCACATTATTGTAGAAAACGCGGACCCACAGCGCACTTTCCAAAGCGGTGCCGCGTCAGAATGCGCTGGCAGAAAAAAATTAATCCAAAAGTACCCTCCAAGCAGCCCATATAAACGCGTTTACAAATCCGCTAACCTCAACAATTTGAGCAGAGAAAATTCGCACCTACAAGGCAGATGGCATCATCATTCAATCCAGAGCAGGCAAGAGTTCCTTCAGCATTACCTTTACCAGCACCACCACTTACCAAATTCAACATCGGACTTTGTCAATTGAGTGTTACTTCTGATAAGAAAAGAAACATTTCACATGCTAAGAAAGCAATCGAAGAGGCTGCTAGTAAGGGAGCTAAACTCGTTCTTTTGCCTGAAATATGGAACTCACCATACAGTAACGATTCTTTTCCTGTGTACGCAGAAGAGATCGATGCTGGAGGTGATGCATCTCCATCAACTGCTATGCTCTCAGAAGTTAGTAAGAGACTCAAGATTACAATTATCGGAGGTTCAATTCCTGAGAGAGTTGGAGATAGGTTGTATAACACATGTTGCGTGTTCGGATCTGATGGAGAGCTCAAGGCTAAGCATAGGAAGATTCACCTCTTCGATATAGATATTCCTGGAAAGATCACCTTCATGGAATCAAAAACACTTACCGCTGGAGAGACTCCAACAATTGTTGATACAGATGTGGGTAGAATCGGAATAGGTATATGTTACGATATCAGGTTCCAAGAATTGGCTATGATATATGCTGCAAGAGGAGCACATCTCTTATGCTACCCTGGAGCTTTCAATATGACTACAGGTCCATTGCACTGGGAGCTTTTGCAAAGAGCTAGGGCAACAGATAACCAGCTCTATGTTGCTACCTGCTCTCCTGCAAGAGATTCAGGAGCTGGTTACACCGCATGGGGTCATTCTACTCTTGTTGGACCATTTGGTGAAGTGTTGGCTACCACTGAGCACGAAGAGGCTATTATAATCGCAGAAATCGATTACAGTATACTTGAGCAGAGAAGGACTTCTCTCCCATTAAATAGGCAGAGGAGGGGTGATTTATACCAGTTAGTTGATGTTCAGAGATTAGATAGTAAGTGACACGTGTGAATTACAGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGGGTACCGACGGGTACCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA

Seedling Inoculations: Alfalfa seedlings were grown to less than about ½inch tall, and were then soaked in paper toweling that had been floodedwith the Agrobacteria containing the TF101.1 vector carrying theΩ-amidase transgene expression cassette. The seedlings were left in thepaper toweling for two to three days, removed and then planted inpotting soil. Resulting TO and control plants were then grown for 27days in a growth chamber, harvested and analyzed for biochemical andphysical characteristics.

Biochemical Characterization: HPLC Assay for 2-oxoglutaramate: HPLC wasused to assay 2-oxoglutaramate, following a modification of Calderon etal., 1985, J Bacteriol 161(2): 807-809. Briefly, 2-oxoglutaramate wasextracted from plant tissue in distilled de-ionized water acidified toless than pH 2.0 with HCl using a weight to volume ratio of 2:1.2-Oxoglutaramate was detected and quantified by HPLC, using an ION-3007.8 mm ID.times.30 cm L column, with a mobile phase in 0.01NH₂SO₄, aflow rate of approximately 0.2 ml/min, at 40.degree. C. Injection volumeis approximately 20 and retention time between about 38 and 39 minutes.Detection is achieved with 210 nm UV light. Authentic 2-oxoglutaramatewas used to calibrate the assay.

HPLC Assays for GPT and GS Activities: GPT was extracted from freshplant tissue after grinding in cold 100 mM Tris-HCl, pH 7.6, containing1 mm ethylenediaminetetraacetic, 200 mM pyridoxal phosphate and 6 mMmercaptoethanol in a ratio of 3 ml per gram of tissue. The extract wasclarified by centrifugation and used in the assay. GS activity wasextracted from fresh plant tissue after grinding in cold 50 mMImidazole, pH 7.5 containing 10 mM MgCl2, and 12.5 mM mercaptoethanol ina ratio of 3 ml per gram of tissue. The extract was clarified bycentrifugation and used in the assay. GPT activity was assayed asdescribed in Calderon and Mora, 1985, Journal Bacteriology 161:807-809.GS activity was measured as described in Shapiro and Stadtmann, 1970,Methods in Enzymology 17A: 910-922. Both assays involve an incubationwith substrates and cofactor at the proper pH. Detection was by HPLC.

HPLC Assay for Ω-Amidase Activity: Ω-amidase activity was determinedusing the 96-well plate assay as described in Krasnikov et al., 2009,Analytical Biochemistry 391: 144-150.

Results:

Plant fresh weight and leaf and root 2-oxoglutaramate concentrations andratios in wild type and transgenic alfalfa plants were measured and areshown in Table 5 below. A comparison of the GS and GPT activities in thebest performing transgenic alfalfa with a wild type control plantaverage values is shown in Table 6. Plant fresh weight values areplotted against 2-oxoglutaramate concentrations in FIG. 4. A photographcomparing transgenic and control alfalfa plants is shown in FIG. 5.

Transgenic alfalfa plants carrying the Ω-amidase directed for rootexpression showed significantly reduced root 2-oxoglutaramateconcentrations, presumably as a result of the added Ω-amidase activityintroduced by the transgene (Table 5). Activity levels for both GS andGPT remain constant in root tissues but are very significantly elevatedin transgenic alfalfa (Table 6).

The transgenic plants also showed faster growth, yielding substantiallyincreased biomass (Table 5), which correlated in near-linear fashionwith the level of reduced 2-oxoglutaramate in these plants (FIG. 4). Thefastest growing transgenic alfalfa line exhibited a 274% increase inbiomass relative to the average biomass of the controls. This line alsoshowed the most reduction in 2-oxoglutaramate root concentration.

TABLE 5 FRESH ROOT LEAF LEAF/ ALFALFA WEIGHT 2-OGM 2-OGM ROOT GENOTYPE(g) (nmol/g) (nmol/g) 2-OGM Control 1 1.43 269.7 910.7 3.5 Control 21.75 286 1536.5 5.4 Control 3 1.16 383.4 1826.6 4.8 Transgene 4 2.31 3322144.6 6.5 Transgene 6 2.80 189 1479.4 7.8 Transgene 13 3.16 241.23038.5 12.8 Transgene 5 5.42 125.14 2729.9 21.8 TG = Trangenic

TABLE 6 GS Activity GPT Activity ALFALFA micromoles/gfwt/minnmoles/gfw/hr GENOTYPE Leaf Root Leaf Root Control. avg 4.3 3.3 339.166.3 Transgene 5 15.9 3.5 551.5 63.6

Example 4 Increased Growth of Transgenic Arabidopsis Plants CarryingRoot-Preferred OMEGA-Amidase Transgene

In this example, Arabidopsis plant growth was increased by introducingan Ω-amidase transgene under the control of a highly root-preferredpromoter. The resulting transgenic Arabidopsis plants showed markedlydecreased 2-oxoglutaramate concentration in roots, very significantlyincreased leaf-to-root ratios of 2-oxoglutaramate, highly elevated GSand GPT levels in leaf, greatly reduced Ω-amidase levels in leaves, andastounding enhanced growth relative to wild type Arabidopsis plants.

Materials and Methods:

Agrobacterium Vectors: The Ω-amidase transgene expression vector andAgrobacteria preparation were generated as described in Example 3,supra.

Transformation: Transformation of Arabidopsis was achieved usingAgrobacterium-mediated “floral dip” transfer as described (Harrison etal., 2006, Plant Methods 2:19-23; Clough and Bent, 1998, Plant J.16:735-743). Agrobacteria transformed with the TF101.1 vector carryingthe 52-amidase transgene expression cassette were grown under antibioticselection, collected by centrifugation resuspended in LB broth withantibiotic and used to floral dip Arabidopsis inflorescence. Floraldipped Arabidopsis plants were taken to maturity and self-fertilized andseeds were collected.

Germination and Selection: Seeds from plants transformed with theTF101.1 vector carrying the Ω-amidase transgene expression cassette weregerminated on a media containing 15 mg/L of BASTA or an equivalentamount of phosphinothricin. For the additional constructs andcombinations described in Examples 5-7: Seeds derived from plantstransformed with glutamine synthetase construct were germinated on amedia contain 20 micrograms hygromycin and regular selection procedureswere followed to obtain the surviving seedlings. Seeds derived fromplants transformed with glutamine phenylpyruvate transaminase weregeminated on a media containing 20 ug/ml of kanamycin and regularselection procedures were followed to obtain the surviving seedlings.For seedlings containing more than one of these genes the seedlings weretransferred to media containing the next selection chemical andsurviving seedlings were obtained. The surviving seedlings wereexamined.

Biochemical Characterization: Assays for 2-oxoglutaramate, Ω-amidase, GSand GPT were conducted as described in Example 3, supra.

Results:

GPT activity and GS activity of wild type and transgenic Arabidopsisplants were measured and are shown in Table 7, below. Ω-amidaseactivities and 2-oxoglutaramate concentrations in wild type andtransgenic Arabidopsis plants were measured in both leaf and roottissues, and are shown in Table 8, below.

TABLE 7 GS Activity GPT Activity Arabidopsis FWt mg umoles/gfwt/minnmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wildtype* 77 1.5 3.4 2.3 167 132 0.8 TG Amidase** 479 1.4 5.8 4.1 192 4862.5 TG = Transgenic *Average values of 9 plants **Average values of 6plants

TABLE 8 2-Oxoglutaramate Ω-amidase Concentration nmoles/gfwt/hrnmoles/gfwt Arabidopsis FWt mg L/R L/R Genotype Whole plant Root LeafRatio Root Leaf Ratio Wild type* 77 86 1090 12.7 410 163 0.4 TG 479 243127 0.5 98 305 3.1 ω-amidase** TG = Transgenic *Average values of 9plants **Average values of 6 plants

Compared to wild type control Arabidopsis plants, the transgenicArabidopsis plants carrying the Ω-amidase directed for root expressionshowed dramatic biochemical changes within the Ω-amidase pathway,including increased root Ω-amidase activity (186% increase), reducedlevels of root 2-oxoglutaramate (76% reduction) and increasedleaf-to-root 2-oxoglutaramate ratios. Moreover, these transgenic plantsalso show great reductions in leaf Ω-amidase activity levels (88%reduction), a near-doubling of leaf 2-oxoglutaramate levels, and higherleaf GS and leaf GPT activities (70% and 533%, respectively). Theresulting impact on growth was astounding, with the transgenic plantsweighing more than six times the weight of the wild type plants, onaverage.

Example 5 Increased Growth of Transgenic Arabidopsis Plants CarryingRoot-Preferred Ω-Amidase Transgene and Leaf-Directed GPT

In this example, Arabidopsis plant growth was increased by introducingan Ω-amidase transgene under the control of a highly root-preferredpromoter and a GPT transgene under the control of a leaf-directingpromoter. The resulting transgenic Arabidopsis plants show astoundingincreases in growth, as well as various biochemical changes, relative towild type Arabidopsis plants.

Materials and Methods:

Agrobacterium Vectors: The Ω-amidase transgene expression vector andAgrobacterium preparation were generated as described in Example 3,supra. For the leaf-directed GPT transgene, an expression cassettecomprising the tomato rubisco small subunit promoter and an Arabidopsiscodon-optimized GPT truncated to delete the first 45 codons of the fulllength GPT (eliminating the chloroplast transit peptide) was constructedand cloned into the Cambia 2201 expression vector. The resulting GPTtransgene expression vector construct (6c) is shown below, and was usedto transform Agrobacteria as described in Example 3.

The nucleotide sequence of the Cambia 2201 with tomato rubisco SSUpromoter+(−45) truncated, optimized for Arabidopsis GPT+nos terminatoris set forth below as SEQ ID NO: 40. Underlined nucleotides=tomatorubisco promoter, bold nucleotides=GPT coding region (codon optimizedfor Arabidopsis), italicized nucleotides=nos terminator region (and someCambia vector sequence), and other nucleotides=Cambia 2201 vector andadditional cloning sites.

CCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGATCTAGAGAATTCATCGATGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTACTTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGCTTCTGAAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCTTCCATTCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCTCCTCCTCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTGATATATCCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGGGTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATGGATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGGAGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTTAGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAGGGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAGCTCTAAAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCATAGGTAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAATACACTAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGTAAGAATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTATAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGACTGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATTATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTGTACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTTTCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATATGGCGCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGGGGTTGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAAATATTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGTGGGTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAATATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGTCAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATTTCAGCAACTAGTATGGCGACTCAAAATGAGTCAACACAAAAGCCTGTTCAGGTGGCTAAGAGACTTGAGAAGTTTAAAACTACAATTTTCACTCAAATGTCTATCCTCGCAGTTAAGCACGGAGCTATTAATCTTGGACAGGGTTTTCCTAACTTCGATGGTCCAGATTTCGTGAAAGAAGCTGCAATTCAAGCAATCAAGGATGGAAAAAATCAGTATGCTAGAGGATACGGTATTCCTCAGTTGAACTCTGCTATCGCTGCAAGATTCAGAGAAGATACAGGACTTGTTGTGGATCCAGAAAAAGAGGTTACTGTGACATCAGGTTGTACTGAGGCTATTGCTGCAGCTATGCTCGGACTTATTAACCCTGGAGATGAAGTTATCCTTTTTGCACCATTCTATGATTCTTACGAGGCTACATTGTCAATGGCAGGAGCTAAGGTGAAAGGTATTACTCTCAGACCTCCAGATTTCTCTATCCCTTTGGAAGAGCTCAAGGCAGCTGTTACTAATAAGACAAGAGCTATCTTGATGAATACTCCTCATAACCCAACAGGAAAGATGTTTACTAGAGAAGAGCTCGAAACTATTGCTTCTCTTTGCATCGAGAACGATGTTTTGGTGTTCTCAGATGAAGTGTATGATAAACTCGCATTTGAGATGGATCACATTTCTATCGCTTCACTTCCAGGAATGTACGAAAGAACTGTTACTATGAATTCTTTGGGAAAGACTTTTTCTCTCACAGGATGGAAAATTGGTTGGGCAATCGCTCCTCCACATCTCACATGGGGTGTTAGACAAGCACACTCTTATCTTACTTTCGCAACTTCAACACCTGCTCAGTGGGCAGCTGTGGCAGCTCTTAAGGCTCCAGAATCTTACTTCAAGGAGTTGAAGAGAGATTACAACGTTAAGAAAGAAACACTTGTGAAGGGATTGAAAGAGGTTGGTTTTACAGTGTTCCCTTCTTCAGGAACTTACTTTGTTGTGGCAGATCATACTCCATTCGGTATGGAAAACGATGTTGCTTTTTGTGAGTATCTTATTGAAGAGGTTGGAGTTGTGGCTATCCCTACATCTGTGTTTTACCTTAATCCAGAAGAGGGAAAGAATCTTGTTAGATTTGCATTCTGCAAAGATGAAGAGACTTTGAGAGGTGCTATTGAGAGGATGAAGCAAAAACTCAAGAGAAAAGTTTGACACGTGTGAATTACAGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAATAATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCTAATCGCTTGAGTTAACGCCGGCGAAGCGGCGTCGGCTTGAACGAATTTCTAGCTAGAGGATCGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATTGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATGATGTTTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACCCCAAAAAAACATGTCATAACAAGAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGACGGCAGTTACGCTACTTGCATTACAGCTTACGAACCGAACGAGGCTTATGTCCACTGGGTTCGTGCCCGAATTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACTCTAGCTAGAGGATCGATCCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGTGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCCCCATGGTCGATCGACAGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCA ATACGCAAACCGCCTCTC

Transformation and Selection: Transformation of Arabidopsis was achievedusing Agrobacterium-mediated “floral dip” transfer as described inExample 4. Transformed plants were grown under selection as described inExample 4.

Biochemical Characterization: Assays for 2-oxoglutaramate, Ω-amidase, GSand GPT were conducted as described in Example 3, supra.

Results:

GPT activity and GS activity of wild type and transgenic Arabidopsisplants were measured and are shown in Table 9, below. Ω-amidaseactivities and 2-oxoglutaramate concentrations in wild type andtransgenic Arabidopsis plants were measured in both leaf and roottissues, and are shown in Table 10, below.

TABLE 9 GS Activity GPT Activity Arabidopsis FWt mg umoles/gfwt/minnmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wildtype* 77 1.5 3.4 2.3 167 132 0.8 TG GPT (6c) + 513 1.8 8.25 4.6 232 3891.7 ω-amidase** TG = Transgenic GPT (6c) = −45 truncated GPT [SEQ ID NO:40] *Average values of 9 plants **Average values of 5 plants

TABLE 10 2-Oxoglutaramate Ω-amidase Concentration nmoles/gfwt/hrnmoles/gfwt Arabidopsis FWt mg L/R L/R Genotype Whole plant Root LeafRatio Root Leaf Ratio Wild type* 77 86 1090 12.7 410 163 0.4 TG GPT(6c) + 513 308 584 1.9 268 275 1.0 ω-amidase ** TG = Transgenic GPT (6c)= −45 truncated GPT [SEQ ID NO: 40] *Average values of 9 plants **Average values of 5 plants

Compared to wild type control Arabidopsis plants, the transgenicArabidopsis plants carrying the Ω-amidase directed for root expressionand the truncated GPT (for cyosolic expression) directed for leafexpression showed biochemical changes within the Ω-amidase pathwaysimilar to those observed in the transgenic plants of Example 5, supra.More specifically, transgenic GPT+Ω-amidase plants showed increased rootΩ-amidase activity, reduced root 2-oxoglutaramate concentration, andincreased leaf-to-root 2-oxoglutaramate ratios. Also, similar to theresults seen in the transgenic plants of Example 5, the GPT+Ω-amidasetransgenic plants showed significant reductions in leaf Ω-amidaseactivity levels, increased leaf 2-oxoglutaramate, and higher leaf GS andleaf GPT activities. The resulting impact on growth was even greaterthan observed for the transgenic plants of Example 5, with thetransgenic plants weighing more than 6.7 times the weight of the wildtype plants, on average.

Example 6 Increased Growth of Transgenic Arabidopsis Plants CarryingRoot-Preferred Ω-Amidase Transgene and Leaf-Directed GPT and GS

In this example, Arabidopsis plant growth was increased by introducingan Ω-amidase transgene under the control of a highly root-preferredpromoter and GPT and GS transgenes under the control of leaf-directingpromoters. The resulting transgenic Arabidopsis plants showed astoundingincreases in growth, as well as various biochemical changes, relative towild type Arabidopsis plants.

Materials and Methods:

Agrobacterium Vectors: The Ω-amidase transgene expression vector andAgrobacteria preparation were generated as described in Example 3,supra. For the leaf-directed GPT transgene, an expression cassettecomprising the tomato rubisco small subunit promoter and the codingsequence for an Arabidopsis codon-optimized GPT truncated to delete thefirst 45 codons of the full length GPT (eliminating the chloroplasttransit peptide), and containing an F to V mutation at amino acidresidue 45, was constructed and cloned into the Cambia 2201 expressionvector. The resulting GPT transgene expression vector construct (9c) isshown in SEQ ID NO: 41 below, and was used to transform Agrobacteria asdescribed in Example 5.

The nucleotide sequence of the Cambia 2201 with tomato rubisco SSUpromoter+(−45) truncated, optimized for Arabidopsis GPT F-to-Vmutation+nos terminator is set forth below as SEQ ID NO: 41. Underlinednucleotides=tomato rubisco promoter, bold nucleotides=GPT coding region(codon optimized for Arabidopsis), italicized nucleotides=nos terminatorregion (and some Cambia vector sequence), and other nucleotides=Cambia2201 vector and additional cloning sites.

CCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGATCTAGAGAATTCATCGATGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTACTTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGCTTCTGAAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCTTCCATTCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCTCCTCCTCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTGATATATCCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGGGTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATGGATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGGAGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTTAGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAGGGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAGCTCTAAAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCATAGGTAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAATACACTAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGTAAGAATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTATAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGACTGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATTATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTGTACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTTTCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATATGGCGCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGGGGTTGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAAATATTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGTGGGTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAATATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGTCAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATTTCAGCAACTAGTATGGCGACTCAAAATGAGTCAACACAAAAGCCTGTTCAGGTGGCTAAGAGACTTGAGAAGTTTAAAACTACAATTTTCACTCAAATGTCTATCCTCGCAGTTAAGCACGGAGCTATTAATCTTGGACAGGGTGTTCCTAACTTCGATGGTCCAGATTTCGTGAAAGAAGCTGCAATTCAAGCAATCAAGGATGGAAAAAATCAGTATGCTAGAGGATACGGTATTCCTCAGTTGAACTCTGCTATCGCTGCAAGATTCAGAGAAGATACAGGACTTGTTGTGGATCCAGAAAAAGAGGTTACTGTGACATCAGGTTGTACTGAGGCTATTGCTGCAGCTATGCTCGGACTTATTAACCCTGGAGATGAAGTTATCCTTTTTGCACCATTCTATGATTCTTACGAGGCTACATTGTCAATGGCAGGAGCTAAGGTGAAAGGTATTACTCTCAGACCTCCAGATTTCTCTATCCCTTTGGAAGAGCTCAAGGCAGCTGTTACTAATAAGACAAGAGCTATCTTGATGAATACTCCTCATAACCCAACAGGAAAGATGTTTACTAGAGAAGAGCTCGAAACTATTGCTTCTCTTTGCATCGAGAACGATGTTTTGGTGTTCTCAGATGAAGTGTATGATAAACTCGCATTTGAGATGGATCACATTTCTATCGCTTCACTTCCAGGAATGTACGAAAGAACTGTTACTATGAATTCTTTGGGAAAGACTTTTTCTCTCACAGGATGGAAAATTGGTTGGGCAATCGCTCCTCCACATCTCACATGGGGTGTTAGACAAGCACACTCTTATCTTACTTTCGCAACTTCAACACCTGCTCAGTGGGCAGCTGTGGCAGCTCTTAAGGCTCCAGAATCTTACTTCAAGGAGTTGAAGAGAGATTACAACGTTAAGAAAGAAACACTTGTGAAGGGATTGAAAGAGGTTGGTTTTACAGTGTTCCCTTCTTCAGGAACTTACTTTGTTGTGGCAGATCATACTCCATTCGGTATGGAAAACGATGTTGCTTTTTGTGAGTATCTTATTGAAGAGGTTGGAGTTGTGGCTATCCCTACATCTGTGTTTTACCTTAATCCAGAAGAGGGAAAGAATCTTGTTAGATTTGCATTCTGCAAAGATGAAGAGACTTTGAGAGGTGCTATTGAGAGGATGAAGCAAAAACTCAAGAGAAAAGTTTGACACGTGTGAATTACAGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAATAATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCTAATCGCTTGAGTTAACGCCGGCGAAGCGGCGTCGGCTTGAACGAATTTCTAGCTAGAGGATCGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATTGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATGATGTTTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACCCCAAAAAAACATGTCATAACAAGAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGACGGCAGTTACGCTACTTGCATTACAGCTTACGAACCGAACGAGGCTTATGTCCACTGGGTTCGTGCCCGAATTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACTCTAGCTAGAGGATCGATCCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACATGATATTCGGCAAGCAGGCATCGCCATGTGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGTGATACTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCCCCATGGTCGATCGACAGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCA ATACGCAAACCGCCTCTC

For the leaf-directed GS transgene, an expression cassette comprisingthe tomato rubisco small subunit promoter and an Arabidopsis GS1 codingsequence was constructed and cloned into the Cambia 1305.1 expressionvector with the tomato rubisco small subunit promoter (rbcS3C). Theresulting GS transgene expression vector construct (4c) is set forth asSEQ ID NO: 43, and was used to transform Agrobacteria as described inExample 5.

The nucleotide sequence of the Cambia 1305.1 with rubisco small subunitpromoter (rbcS3C)+ARGS (Arabidopsis GS1) is set forth below as SEQ IDNO: 43. Underlined nucleotides=tomato rubisco promoter, doubleunderlined nucleotides=catI intron in the Cambia 1305.1 vector first 10amino acids are from GUSplus enzyme and cloning sites in 1305.1 vector,bold nucleotides=Arabidopsis GS1 coding region (cloned into SpeI to pmIIsites), italicized nucleotides=nos terminator region (and some Cambiavector sequence), and other nucleotides=Cambia 2201 vector andadditional cloning sites.

GGTACCGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTACTTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGCTTCTGAAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCTTCCATTCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCTCCTCCTCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTGATATATCCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGGGTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATGGATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGGAGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTTAGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAGGGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAGCTCTAAAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCATAGGTAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAATACACTAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGTAAGAATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTATAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGACTGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATTATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTGTACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTTTCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATATGGCGCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGGGGTTGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAAATATTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGTGGGTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAATATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGTCAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATTTCAGCACCATGGTAGATCTGAGGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAGAACCGACGAACTAGTATGTCTCTGCTCTCAGATCTCGTTAACCTCAACCTCACCGATGCCACCGGGAAAATCATCGCCGAATACATATGGATCGGTGGATCTGGAATGGATATCAGAAGCAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCAAAGCTTCCCAAGTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGAAGACAGTGAAGTCATTCTATACCCTCAGGCAATATTCAAGGATCCCTTCAGGAAAGGCAACAACATCCTGGTGATGTGTGATGCTTACACACCAGCTGGTGATCCTATTCCAACCAACAAGAGGCACAACGCTGCTAAGATCTTCAGCCACCCCGACGTTGCCAAGGAGGAGCCTTGGTATGGGATTGAGCAAGAATACACTTTGATGCAAAAGGATGTGAACTGGCCAATTGGTTGGCCTGTTGGTGGCTACCCTGGCCCTCAGGGACCTTACTACTGTGGTGTGGGAGCTGACAAAGCCATTGGTCGTGACATTGTGGATGCTCACTACAAGGCCTGTCTTTACGCCGGTATTGGTATTTCTGGTATCAATGGAGAAGTCATGCCAGGCCAGTGGGAGTTCCAAGTCGGCCCTGTTGAGGGTATTAGTTCTGGTGATCAAGTCTGGGTTGCTCGATACCTTCTCGAGAGGATCACTGAGATCTCTGGTGTAATTGTCAGCTTCGACCCGAAACCAGTCCCGGGTGACTGGAATGGAGCTGGAGCTCACTGCAACTACAGCACTAAGACAATGAGAAACGATGGAGGATTAGAAGTGATCAAGAAAGCGATAGGGAAGCTTCAGCTGAAACACAAAGAACACATTGCTGCTTACGGTGAAGGAAACGAGCGTCGTCTCACTGGAAAGCACGAAACCGCAGACATCAACACATTCTCTTGGGGAGTCGCGAACCGTGGAGCGTCAGTGAGAGTGGGACGTGACACAGAGAAGGAAGGTAAAGGGTACTTCGAAGACAGAAGGCCAGCTTCTAACATGGATCCTTACGTTGTCACCTCCATGATCGCTGAGACGACCATACTCGGTTGACACGTGTGAATTGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACAGATCCGGTCGGCATCTACTCTATTTCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACACAGCCATCGGTCCAGACGGCCGCGCTTCTGCGGGCGATTTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGTCGTGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGCTGCTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAATCCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCATGTAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGATCGGCCGCAGCGATCGCATCCATAGCCTCCGCGACCGGTTGTAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGTGCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTAAACTCCCCAATGTCAAGCACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGATAAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGGACATATCCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGAGAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAGTTCAGGCTTTTTCATATCTCATTGCCCCCCGGGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTC

Transformation and Selection: Transformation of Arabidopsis was achievedusing

Agrobacterium-mediated “floral dip” transfer as described in Example 4.Transformed plants were grown under selection as described in Example 4.

Biochemical Characterization:

Assays for 2-oxoglutaramate, Ω-amidase, GS and GPT were conducted asdescribed in Example 3, supra.

Results:

GPT activity and GS activity of wild type and transgenic Arabidopsisplants were measured and are shown in Table 11, below. Ω-amidaseactivities and 2-oxoglutaramate concentrations in wild type andtransgenic Arabidopsis plants were measured in both leaf and roottissues, and are shown in Table 12, below.

TABLE 11 GS Activity GPT Activity Arabidopsis FWt mg umoles/gfwt/minnmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wildtype* 77 1.5 3.4 2.3 167 132 0.8 TG GPT (9c) + 709 1.6 9.4 5.9 182 4142.3 GS (4c) + ω-amidase** TG = Transgenic GPT (9c) = −45 truncated GPTvariant [SEQ ID NO: 41] GS 4c = SEQ ID NO: 43 *Average values of 9plants **Average values of 5 plants

TABLE 12 2-Oxoglutaramate Ω-amidase Concentration nmoles/gfwt/hrnmoles/gfwt Arabidopsis FWt mg L/R L/R Genotype Whole plant Root LeafRatio Root Leaf Ratio Wild type* 77 86 1090 12.7 410 163 0.4 TG GPT(9c) + 709 195 344 1.8 113 223 2.0 GS (4c) + ω-amidase ** TG =Transgenic GPT (9c) = −45 truncated GPT variant [SEQ ID NO: 41] *Averagevalues of 9 plants ** Average values of 5 plants

Compared to wild type control Arabidopsis plants, the transgenicArabidopsis plants carrying the Ω-amidase directed for root expression,and truncated GPT (for cyosolic expression) and GS1 directed for leafexpression, showed biochemical changes within the Ω-amidase pathwaysimilar to those observed in the transgenic plants of Examples 4 and 5,supra. More specifically, transgenic GPT+Ω-amidase plants showedincreased root Ω-amidase activity, reduced root 2-oxoglutaramateconcentration, and increased leaf-to-root 2-oxoglutaramate ratios. Also,similar to the results seen in the transgenic plants of Examples 5 and6, the GPT+GS+Ω-amidase transgenic plants showed significant reductionsin leaf Ω-amidase activity levels, increased leaf 2-oxoglutaramate, andhigher leaf GS and leaf GPT activities. The resulting impact on growthwas greater than observed for the transgenic plants of either Example 4or Example 5, with the transgenic plants weighing more than nine timesthe weight of the wild type plants, on average.

Example 7 Comparison of Transgenic Arabidopsis Genotypes CarryingRoot-Preferred Ω-Amidase Transgene

The data generated from the studies of Examples 4, 5, and 6, which wereconducted in parallel, are presented together for comparison in Tables13 and 14 below.

TABLE 13 GS Activity GPT Activity FWt mg umoles/gfwt/min nmoles/gfwt/hrGenotype Whole plant Root Leaf L/R Root Leaf L/R Wild type* 77 1.5 3.42.3 167 132 0.8 WT + 479 1.4 5.8 4.1 192 486 2.5 ω-amidase** GPT (6c) +513 1.8 8.25 4.6 232 389 1.7 ω-amidase GPT (9c) + 709 1.6 9.4 5.9 182414 2.3 GS + ω-amidase *** *Average values of 9 plants **Average valuesof 6 plants *** Average values of 5 plants

TABLE 14 2-Oxoglutaramate Ω-amidase Concentration FWt mg nmoles/gfwt/hrnmoles/gfwt Genotype Whole plant Root Leaf L/R Root Leaf L/R Wild type*77 86 1090 12.7 410 163 0.4 WT + 479 243 127 0.5 98 305 3.1 ω-amidase **GPT (6c) + 513 308 584 1.9 268 275 1.0 ω-amidase *** GPT (9c) + 709 195344 1.8 113 223 2.0 GS + ω-amidase *** *Average values of 9 plants **Average values of 6 plants *** Average values of 5 plants

Example 8 Chemical Inhibition of Ω-Amidase Activity in Leaf and RootTissues and Modulation of Leaf-to-Root Ratio of 2-Oxoglutaramate

This example demonstrates how the concentration of 2-oxoglutaramatechanges in response to foliar and root treatment with6-diazo-5-oxo-nor-leucine (DON), an inhibitor of the Ω-amidase enzymewhich breaks-down 2-oxoglutaramate (Duran and Calderon, 1995, Role ofthe glutamine transaminase-Ω-amidase pathway and glutaminase inglutamine degradation in Rhizobium etli. Microbiology 141:589-595).

Materials and Methods:

Nutrient Solution:

Columbia nutrient solution was utilized (Knight and Weissman, 1982,Plant Physiol. 70: 1683).

Leaf Treatments:

Plants were grown in sand at 24.degree. C. using a 16 hour/day light and8 hour/day dark photoperiod. Seeds were germinated in the sand andallowed to grow for 9-14 days after seedling emergence. Seedlings (1 per3 inch pot) were provided nutrients daily. The top of each pot wascovered with Saran plastic film, with a slit cut to allow the seedlingsroom to emerge, in order to prevent treatment solution from reaching thesoil. Leaves were treated by spraying DON treatment/nutrient solutiontwice daily. The DON treatment/nutrient solution contained 1microgram/ml DON, 50 micoliters/liter SILWET L77 (a surfactant) and 0.02vol/vol % glycerol (a humectant) at pH 6.3, dissolved in nutrientsolution. Control plants were sprayed daily with the same solutionwithout DON. An airbrush was used to apply the solutions until drip.Plants were sacrificed and analyzed for fresh weight, Ω-amidaseactivity, and 2-oxoglutaramate concentrations in leaf and root tissue 14days after initiation of treatment.

Root Treatments:

Plants were grown hydroponically at 24.degree. C. using a 16 hour/daylight and 8 hour/day dark photoperiod. Seeds were first germinated andseedlings were grown for 9 days, at which time the individual seedlingswere suspended with their roots in the nutrient solution (600 ml ofnutrient, pH 6.3) in an 800 ml beaker covered with aluminum foil, with aslit for the seedlings, to prevent algal growth. The beakers wereaerated with air provided by a small pump and delivered into thesolution through a glass Pasteur pipette. For the treatments, DON wasadded to the nutrient solution to a final concentration of 1 microgramper ml. Thus DON was supplied continuously to the treated seedlings. Thecontrols were grown in the same nutrient solution without DON. Thesolutions were refreshed every third day. Plants were sacrificed andanalyzed for fresh weight, Ω-amidase activity, and 2-oxoglutaramateconcentrations in leaf and root tissue 14 days after initiation oftreatment.

Results:

Treatment of Roots with Ω-Amidase Inhibitor:

Sweet corn and pole bean plant roots were treated with DON as describedabove. The results of the root treatments are shown in Tables 15 and 16below. None of the DON treated plants had detectable levels of Ω-amidaseactivity, whereas the control wild type plants maintained normal levelsof Ω-amidase activity. Indeed, all corn and bean plants subjected tocontinuous DON treatment of roots showed severely stunted growth, incontrast to vigorous growth of untreated control plants. Control plantsincreased their fresh weighs throughout the experimental period up tonearly eight-fold.

Dramatic reductions in the 2-oxoglutaramate leaf-to-root ratio wereobserved in both corn and bean plants treated with the Ω-amidaseinhibitor. Treated plants accumulated very large amounts of2-oxoglutaramate in their roots (over 30-fold increase in corn; 15-foldincrease in beans) while maintaining normal levels in their leaves.

TABLE 15 INHIBITION OF Ω-AMIDASE IN CORN ROOTS % INCREASE 2-OGM 2-OGMLEAF/ IN FWT LEAF ROOT ROOT CORN (initial wt) nmoles/gfwt nmoles/gfwtRATIO CONTROL 370% 275 65 4.25 (1.26 g) TREATED 174% 300 2286 0.13 (0.96g) 2-OGM = 2-oxoglutaramate

TABLE 16 INHIBITION OF Ω-AMIDASE IN BEAN ROOTS % INCREASE 2-OGM 2-OGMLEAF/ IN FWT LEAF ROOT ROOT BEAN (initial wt) nmoles/gfwt nmoles/gfwtRATIO CONTROL 839% 311.2 101.4 3.07 (1.134 g) TREATED 198% 279 1586 0.18(1.104 g) 2-OGM = 2-oxoglutaramate

Treatment of Leaves with Ω-Amidase Inhibitor:

Corn plant leaves were treated with DON as described above. The resultsare shown in Tables 17 and 18, below. Ω-amidase activity in leaf waseffectively suppressed by DON, with the treated plants showing onlyabout 50% the Ω-amidase activity observed in untreated plants (Table18). A dramatic increase in leaf 2-oxoglutaramate and the leaf-to-root2-oxoglutaramate ratio was observed in the treated plants (Tables 17 and18). Specifically, treated plants accumulated very large amounts of2-oxoglutaramate in their leaves (more than 7-fold increase overuntreated plants). Moreover, inhibition of Ω-amidase activity in leavesalso resulted in a near doubling of corn plant fresh weights (Table 18).

TABLE 17 INHIBITION OF Ω-AMIDASE IN CORN LEAVES RESULTS IN INCREASEDLEAF-TO-ROOT RATIO 2-OXOGLUTARAMATE 2-OGM LEAF 2-OGM ROOT LEAF/ROOT CORNnmoles/gfwt nmoles/gfwt RATIO CONTROL 101.3 344.1 0.29 TREATED 753.9126.8 5.0 2-OGM = 2-oxoglutaramate gfwt = grams fresh weight

TABLE 18 INHIBITION OF Ω-AMIDASE IN CORN LEAVES RESULTS IN INCREASEDGROWTH 2-Oxoglutaramate Whole Plant Fresh Amidase activity Leaf,Concentration Wt, g μmole/gfw/hr nmoles/gfwt CORN Control TreatedControl Treated Control Treated 9.3 20.9 12.4 19.2 11.3 24.3 Average11.0 21.4 0.261 0.135 101.3 838.8 gfwt = grams fresh weight

Example 9 Comparison of GPT Isoforms in Combination with Root-PreferredΩ-Amidase

This Example compares the growth-enhancing performance of threedifferent GPT transgene isoforms in combination with root-preferredexpression of an Ω-amidase transgene on Arabidopsis plant growth. TheΩ-amidase expression construct used in all three combinations is asdescribed in Example 3 [SEQ ID NO: 39]. The three GPT transgene isoformswere: (1) GPT 5c, full length Arabidopsis GPT codon optimized [SEQ IDNO: 42]; (2) GPT 6c, truncated −45 GPT (deleted chloroplast targetingsequence), codon optimized [SEQ ID NO: 40]; and, (3) GPT 9c, truncated−45 GPT (deleted chloroplast targeting sequence), codon optimized,mutation F to V at amino acid residue 45 [SEQ ID NO: 41].

The nucleotide sequence of the Cambia 1305.1 with rbcS3C promoter+catIintron with Arabidopsis GPT gene is set forth below as SEQ ID NO: 42.Underlined ATG is start site, parentheses are the catI intron and theunderlined actagt is the speI cloning site used to splice in theArabidopsis gene.

AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)AACCGACGA ATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTAGCTTCAAAGCCTCTCTTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAAAATCCATCGTCCTATCGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTCTACTCAAAAACCCGTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATGAGCATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTACATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTATGAACACTCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAAGCACACTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGCAGAAGCTTAAGAGAAAAGT  CTGA

The results are shown in Table 19 below. Both of the GPT isoforms inwhich the chloroplast transit peptide was deleted outperform the wildtype and full length GPT isoform plants.

TABLE 19 TRUNCATED GPT ISOFORMS OUTPERFORM NATIVE GPT Fresh Mean %Genotype Wt, mg weight, mg SD+/− Increase Wild type (average of 77 27 09 plants 5c GFT + ω-amidase 357 456 459 198 371 149 482 6cGPT +ω-amidase 525 438 560 501 552 513 56 666 9c GPT + ω-amidase 390 359 405387 574 431 99 560

1-41. (canceled)
 42. A method for generating transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production to thereby improve growth characteristics of the transgenic plants, comprising: (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein the Ω-amidase transgene is operably linked to a heterologous promoter; (b) generating a transgenic plant from the plurality of plant cells; and (c) expressing the Ω-amidase transgene in root tissue of the transgenic plant or the progeny of the transgenic plant, wherein said transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramate production relative to an analogous wild type or untransformed plant.
 43. The method of claim 42, wherein the Ω-amidase transgene codes for a polypeptide having Ω-amidase catalytic activity and an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:
 38. 44. The method according to claim 42, wherein the Ω-amidase transgene is incorporated into the genome of the plant.
 45. The method according to claim 42, wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S²promoter, GI4 promoter, GI5 promoter, and GRP promoter.
 46. The method according to claim 42, wherein endogenous Ω-amidase expression in leaf tissue is inhibited.
 47. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor.
 48. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected from the group consisting of a mutant Ω-amidase gene that eliminates endogenous Ω-amidase expression, an endogenous Ω-amidase knockout mutant, and an endogenous Ω-amidase knockdown mutant.
 49. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous Ω-amidase gene.
 50. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from the group consisting of 6-diazo-5-oxonor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
 51. The method according to claim 42, wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type plant or untransformed plant.
 52. The method according to claim 42, wherein the transgenic plant further comprises a GPT transgene.
 53. The method according to claim 52, wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1.
 54. The method according to claim 42, wherein the transgenic plant further comprises a GPT transgene and a GS transgene.
 55. The method according to claim 54, wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
 56. The method according to claim 42, wherein endogenous GPT expression in the transgenic plant is increased by gene activation.
 57. The method according to claim 42, wherein endogenous GS expression in the transgenic plant is increased by gene activation.
 58. The method according to claim 42, wherein the Ω-amidase transgene is codon optimized for expression in the plant. 59-72. (canceled)
 73. The method according to claim 42, wherein said transgenic plant or said progeny of said transgenic plant has at least one increased growth characteristic selected from the group consisting of increased biomass yield, increased NO₃ uptake, increased chlorophyll per unit weight, increased CO₂ fixation, and increased seed yield.
 74. The method according to claim 42, further comprising selecting a progeny said transgenic plant having increased growth characteristic relative to an analogous wild type or untransformed plant, wherein the growth characteristic is selected from the group consisting of increased biomass yield, increased NO₃ uptake, increased chlorophyll per unit weight, increased CO₂ fixation, and increased seed yield.
 75. The method according to claim 42, wherein the plant is a monocotyledonous plant.
 76. The method according to claim 42, wherein the plant is a dicotyledonous plant.
 77. The method according to claim 42, wherein the plurality of plant cells are taken from a plant selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
 78. The method according to claim 42, wherein the promoter is a root-preferred promoter.
 79. The method according to claim 42, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence according to SEQ ID NO:
 3. 80. The method according to claim 42, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
 3. 81. The method according to claim 42, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:
 3. 82. A progeny of any generation of the transgenic plant generated according to claim 42, wherein the progeny comprises the Ω-amidase transgene.
 83. A seed of any generation of the transgenic plant generated according to claim 42, wherein the progeny comprises the Ω-amidase transgene.
 84. A method for generating and selecting transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production and improved growth characteristics of the transgenic plants, comprising: (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein said Ω-amidase transgene includes a heterologous plant promoter; (b) generating a plurality of transgenic plants from the plant cells; (c) expressing the Ω-amidase transgene in root tissue of the plant or the progeny of the plant; and (d) selecting a transgenic plant having an increased leaf-to-root ratio of 2-oxo-glutaramate production relative to an analogous wild type or untransformed plant.
 85. The method according to claim 84, wherein said transgenic plant or said progeny of the transgenic plant has at least one increased growth characteristic selected from the group consisting of increased biomass yield, increased NO₃ uptake, increased chlorophyll per unit weight, increased CO₂ fixation, and increased seed yield.
 86. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence according to SEQ ID NO:
 3. 87. The method according to claim 84, wherein the heterologous plant promoter is a root-preferred promoter.
 88. The method according to claim 87, wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S²promoter, GI4 promoter, GI5 promoter, and GRP promoter.
 89. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having Ω-amidase catalytic activity and an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:
 38. 90. The method according to claim 84, wherein the Ω-amidase transgene is incorporated into the genome of the plant.
 91. The method according to claim 84, wherein the plant is a monocotyledonous plant.
 92. The method according to claim 84, wherein the plant is a dicotyledonous plant.
 93. The method according to claim 84, wherein the plurality of plant cells are taken from a plant selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
 94. The method according to claim 84, wherein said transgenic plant produces more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant.
 95. The method according to claim 84, wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant.
 96. The method according to claim 84, wherein the transgenic plant has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant.
 97. The method according to claim 84, wherein the plant further comprises a GPT transgene.
 98. The method according to claim 97, wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1.
 99. The method according to claim 84, wherein the plant further comprises a GPT transgene and a GS transgene.
 100. The method according to claim 99, wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
 101. The method according to claim 84, wherein endogenous GPT expression in the plant is increased by gene activation.
 102. The method according to claim 84, wherein endogenous GS expression in the plant is increased by gene activation.
 103. The method according to claim 84, wherein the transgene is codon optimized for expression in the plant.
 104. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
 3. 105. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:
 3. 106. A method for generating and selecting transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production and increased growth characteristics of the transgenic plants, comprising: (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein said Ω-amidase transgene includes a heterologous plant promoter; (b) generating a plurality of transgenic plants from the plant cells; and (c) selecting from said plurality of transgenic plants a transgenic plant having an increased biomass yield relative to an analogous wild type or untransformed plant, wherein a polypeptide encoded by said Ω-amidase transgene catalyzes the synthesis of 2-oxo-glutaramate.
 107. The method according to claim 106, wherein said transgenic plant or said progeny of the transgenic plant has at least one additional increased growth characteristic selected from the group consisting of increased NO₃ uptake, increased chlorophyll per unit weight, increased CO₂ fixation, and increased seed yield.
 108. The method according to claim 106, wherein said polypeptide has an amino acid sequence according to SEQ ID NO:
 3. 109. The method according to claim 106, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:
 3. 110. The method according to claim 106, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:
 3. 111. The method according to claim 106, wherein the heterologous plant promoter is a root-preferred promoter.
 112. The method according to claim 111, wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S²promoter, GI4 promoter, GI5 promoter, and GRP promoter.
 113. The method according to claim 106, wherein said polypeptide has an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO:
 38. 114. The method according to claim 106, wherein the Ω-amidase transgene is incorporated into the genome of the transgenic plant.
 115. The method according to claim 106, wherein the transgenic plant is a monocotyledonous plant.
 116. The method according to claim 106, wherein the transgenic plant is a dicotyledonous plant.
 117. The method according to claim 106, wherein the transgenic plant is selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
 118. The method according to claim 106, wherein said transgenic plant produces more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant.
 119. The method according to claim 106, wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant.
 120. The method according to claim 106, wherein the transgenic plant has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant.
 121. The method according to claim 106, wherein the transgenic plant further comprises a GPT transgene.
 122. The method according to claim 121, wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1.
 123. The method according to claim 106, wherein the transgenic plant further comprises a GPT transgene and a GS transgene.
 124. The method according to claim 123, wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
 125. The method according to claim 106, wherein endogenous GPT expression in the transgenic plant is increased by gene activation.
 126. The method according to claim 106, wherein endogenous GS expression in the transgenic plant is increased by gene activation.
 127. The method according to claim 106, wherein the Ω-amidase transgene is codon optimized for expression in the plant. 